PND01 STANDARD OPERATING PROCEDURE FOR COMPUTER

PND01 STANDARD OPERATING PROCEDURE FOR COMPUTER
STANDARD OPERATING PROCEDURE FOR COMPUTER SUPPLIES AND
EQUIPMENT
CODE: PND01
Effective Date:
2
19
2008
1.
PURPOSE: To describe the minimal requirements for computer hardware and software needed for analysis of
PulseNet PFGE gels.
2.
SCOPE: This procedure applies to all PulseNet participating laboratories analyzing PFGE gels.
3.
DEFINITIONS/TERMS:
3.1 PFGE: Pulsed-field Gel Electrophoresis
3.2 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
3.3 CDC: Centers for Disease Control and Prevention
4.
RESPONSIBILITIES/PROCEDURE:
4.1 Participating laboratories must use the following software and hardware
4.1.1
BioNumerics Software v3.0, v3.5, v4.01, or v5.0
4.1.2
CDC PulseNet masterscripts
4.1.3
Personal computer equipped with an Intel Pentium CPU or better, 256 MB RAM or more, 65 K
color graphics or better and Windows 98, Windows NT 4.0, or higher
4.1.4
Keyboard, mouse, CD-ROM
4.1.5
High resolution screen 1024x768 or higher; true color
4.1.6
Large screen recommended (17" or more)
4.2 For quotes and additional information in USA and Canada, contact Applied Maths, Inc.
4.3 For quotes and additional information in Europe, contact Applied Maths BVBA.
Use of trade names and commercial sources is for identification only and does not imply endorsement by CDC or
the U.S. Department of Health and Human Services.
5.
FLOW CHART:
6.
BIBLIOGRAPHY:
7.
CONTACTS:
7.1 Applied Maths, Inc.
512 East 11th Street, Suite 207
Austin, TX 78701
Phone: 512-482-9700
Fax: 512-482-9708
E-mail: info-US@applied-maths.com
www.applied-maths.com
7.2 Applied Maths BVBA
Keistraat120
B-9830 Sint-Martens-Latem
Belgium
Phone: +32-9-2222-100
Fax: +32-9-2222-102
E-mail: info@applied-maths.com
www.applied-maths.com
8.
AMENDMENTS:
VERSION:
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AUTHORIZED BY:
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STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS
Installation and Use of BioNumerics Gel Analysis Software for PulseNet
Participants
CODE: PND02
5
Effective Date:
9
05
1. PURPOSE : To describe the guidelines for standardization of TIFF image analysis in order to accurately
compare PFGE patterns between PulseNet participating laboratories.
2. SCOPE: This procedure applies to all analyses submitted to PulseNet, thereby allowing comparison of results
with other PulseNet laboratories.
3. DEFINITIONS/TERMS:
3.1
3.2
3.3
3.4
SOP: Standard Operating Procedure
PFGE: Pulsed-field Gel Electrophoresis
TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics
BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
4. RESPONSIBILITIES:
4.1 After an electronic image of a gel is obtained using the procedure in SOP PNL07, the file MUST be
analyzed using the PulseNet customized version of BioNumerics Analysis Software version 3.0 or higher.
4.2 The SOPs PNG05, PNL01 – PNL06 and PND01 contain settings and instructions to allow users to create
and compare analyzed PFGE fingerprint patterns sent to the PulseNet national databases. Masterscripts for
setting up databases in E. coli, Listeria, Salmonella, Shigella, and Campylobacter are available from the
PulseNet Task Force by sending an e-mail to pfge@cdc.gov. In order for results to be compatible with the
PulseNet national databases, databases must be set up using the masterscripts provided by the PulseNet
Task Force.
4.3 TIFFs can either be copied to the appropriate images directory or imported directly into BioNumerics.
4.4 Local labs can add information fields to their databases; however, they should add their lab ID either to the
beginning or end of the information field name. This is recommended to ensure that the local labs are able
to differentiate between CDC fields and their local lab fields.
4.5 When creating a bundle file to be shared with other PulseNet participating labs, and/or on the WebBoard,
the “PulseNet Bundle” tool must be used.
5.
PROCEDURE:
5.1 Installing BioNumerics Software
5.1.1
Insert the BioNumerics CD into the CD-ROM drive and click on “Install BioNumerics.”
5.1.2
Note the directory the installation will be in and use the default setting to install a sample
database (DemoBase).
5.1.3
Click “Yes” to create a shortcut icon
5.1.4
Click “OK” for the protection key installer
5.1.5
Click “Next” to install the sentinel system driver
5.1.6
Accept the terms and click “Next”
5.1.7
Choose the default folder and then click “Next”
5.1.8
Choose the default “Complete” setup type and then click “Next”
5.1.9
Clink “Install” to begin the installation process
5.1.10 Insert the license string, which is found in the BioNumerics package
5.1.11 Click “Finish”
5.2 Creating a new database
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STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS
Installation and Use of BioNumerics Gel Analysis Software for PulseNet
Participants
5.2.1
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Double-click on the BioNumerics icon and click the “New” button.
When the new database window appears, enter a name for the new database and click
“Next.”
Choose which directory you would like to install the database into. Click “Next” to accept the
default directory or change the directory path by using the browse key, and then click “Next.”
When asked if you would like to create log files for the new database, select “Yes” and click the
“Finish” button. The log files allow you to track any changes made to the database.
5.3 Running masterscripts
5.3.1
Insert the most recent PulseNet masterscripts CD (provided by PulseNet) into the CD-ROM drive.
5.3.2
Start the BioNumerics software, highlight the database name that you wish to customize for
PulseNet and click the “Analyze” button.
5.3.3
In the upper toolbar click “Scripts” and select “Run script from file…”
5.3.4
Choose “Install.BNS” from the PulseNet Master Scripts CD.
5.3.5
Select the appropriate organism and enter your lab ID; then click “OK.”
5.3.6
The program will ask to be restarted, click “OK” to finish the installation of Master Scripts.
5.4 Changing database settings
5.4.1
Highlight the database of interest and click “Settings.”
5.4.2
By clicking on the tabs, various settings can be changed, including the background color and the
order of the database fields.
5.5 Importing a TIFF into BioNumerics
5.5.1
Highlight the database of interest and click “Analyze.”
5.5.2
Right-click under the “Files” panel in the main BioNumerics screen and click on “Add new
experiment file…,” or click on the folder shortcut button at the top of the Files menu.
5.5.3
“Browse,” and choose the location of the TIFF, highlight it, and click “Open.”
5.5.4
In BioNumerics v4.0, a confirmation window will ask, “Do you want to edit the image before
adding it to the database?”
5.5.4.1 Choose “No” if you do not need to edit the image.
5.5.4.2 If you choose “Yes,” you can rotate or crop the image, or invert colors.
5.5.5
There should now be a red “N” in front of the TIFF name (the “N” denotes a new TIFF that has
not been analyzed).
5.6 Saving a TIFF into an organism-specific BioNumerics database from an e-mail or WebBoard
5.6.1
When you save a TIFF to analyze in BioNumerics, you will need to save it in a specific file
location.
5.6.2
Save the TIFF in the “Images” folder in the BioNumerics directory for the specified organism. For
example, to save a TIFF in the E. coli BioNumerics database, you would save it under:
C:\Program Files\BioNumerics\data\Ecoli\Images.
5.6.3
The next time the specified database is open, the TIFF will appear under the “Files” panel with a
red “N” in front of it.
5.6.4
Note: The TIFF will not appear if it is saved in the “Images” folder while BioNumerics is open.
To see the newly imported TIFF, close, then reopen BioNumerics.
5.7 Analyzing a gel
5.7.1
Locate the TIFF you wish to analyze in the “Files” area and double-click on the file name, or
highlight the TIFF name and click on the blue arrow shortcut button.
5.7.2
When the “Fingerprint File” window opens, click on the “Edit fingerprint data” button.
5.7.3
Select the appropriate fingerprint type (this will be the enzyme used for the majority of the gel)
and click “OK.”
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STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS
Installation and Use of BioNumerics Gel Analysis Software for PulseNet
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The gel should be white with black bands. If the gel appears black with white bands, go to “Edit
settings” and check the box for “Inverted values.”
Once the Fingerprint data window opens, the selected TIFF image appears in a green box.
Using the green nodes, adjust the size of the green box so that the left and right sides of the green
box are at the edge of the left and rightmost gel lanes. Adjust the top of the green box so that it is
just below the wells of the gel. Position the bottom of the green box at the bottom of the gel or the
bottom of the TIFF (if the bottom of the gel is not visible).
If the gel is curved, holding down the <Shift> key while adjusting the nodes will allow for the box
to curve around the gel.
Next, indicate the lanes on the gel by clicking on the “Auto search lanes” button (magnifying
glass) to perform a lane search.
The Search lanes window will appear and prompt you for an estimated number of lanes on the gel.
This number doesn’t have to be exact but it should be close to the number of lanes since this will
help the program to designate the correct number of lanes. Enter the number of lanes and click
“OK.” Each lane that encompasses the DNA fingerprint is termed a “Gel Strip.”
If the exact number of lanes is known, you may choose to add them by choosing “Lanes” and
“Define group of lanes…” and entering the exact number of lanes on the gel.
When the lane auto search is complete, the “Gel Strip” may need to be adjusted to encompass the
entire PFGE pattern. Blue nodes located inside of the “Gel Strips” allow these lane indicators to be
moved and curved to match the lanes on the gel. To move the entire gel strip, click on any node
and move the gel strip left or right. To adjust the curvature of a gel strip, hold down the <Shift>
key and click on a node and drag it (hold down the mouse button while you move it) to curve the
gel strip. How the gel strip is curved depends on the selected node.
Further adjustments to the fingerprint conversion can be made in the Fingerprint conversion
settings window. Click on the “Edit Settings” button or choose “Edit” then “Edit Settings” from
the top menu to open this window. From this window you can adjust the thickness of the image
gel strips so that they fit the gel lanes. You can also adjust the number of nodes in the lane
indicators to allow for more adjustment flexibility. When you are done with adjustments click
“OK” to close the window and apply the changes.
Once the gel strips have been placed and adjusted to your satisfaction, adjustments to the
appearance of the gel image can be made in the “Gel tone curve” window. To open the window,
click on the “Edit” menu and click on “Edit tone curve.” The “Gel tone curve” window has a set of
image adjustment buttons and a pair of gel lane windows that show a “before” and “after” preview
of any adjustments before they are applied. Furthermore, by clicking in the bottom preview
window and dragging the mouse, the gel image can be moved around within the “after” window.
The “Linear” adjustment on the “Gel tone curve” window is a good starting point to adjust the
color of a gel image. This adjustment selects for the “best fit” Optical Density (OD) range or
densitometric curve values for optimizing the TIFF image. To use this function click on the
“Linear” button. Other adjustments that can be used to improve a gel image are “Enhance weak
bands” and “Enhance dark bands.” These two functions are complementary, so using one and then
the other will cancel any effect. Click on the “Enhance weak bands” button to apply this
adjustment. The button can be clicked a number of times to increase the level of adjustment.
When satisfied with all tone curve adjustments click “OK” to accept the changes (or click
“Cancel” to reject any changes), which will close the “Gel tone curve” window.
When you are satisfied with the gel strip positions and any image adjustments that have been
made, click on the “Next step” button (right arrow) to proceed to Step 2 of Analysis.
The goal of Step 2 is to place the Curve strips so that they are located in the area of the lane that
gives the most accurate representation of the gel bands. The densitometric curve for the selected
lane is shown on the right side of the window. The curve peaks indicate where the gel lane is
“darkest” or has the highest optical density. Click on the nodes in the Curve strips and drag the
mouse to move the whole strip from left to right. By holding down the <Shift> key and clicking
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STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS
Installation and Use of BioNumerics Gel Analysis Software for PulseNet
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on a node, the Curve strip can be curved. You should avoid placing the curve strip in areas with
artifacts (specks, etc.) on the gel.
Once the Curve strips have been successfully placed, click on the “Next Step” button to go to Step
3 of Analysis, which is a step for Normalization where the gel lanes are normalized against the
PulseNet global standard.
Note: During any step of analysis, you can save your work, undo and redo actions, and zoom in or
out by clicking on the various buttons in the upper toolbar.
Step 3, “Normalization,” allows laboratories to compare PFGE results and is the most important
step of analysis. Click on a reference lane to select it (the number above the lane should turn
yellow) and click on the “Use selected lane as reference lane” button (looks like a weight). This
will mark the lane as a reference lane. Repeat this process for all of the reference lanes.
Match the bands in the reference lanes from the gel strips with the global standard band markers
(flags with fragment sizes on them).
To mark a gel strip reference lane band, first select the desired lane by clicking on it. The gel strip
number turns yellow when selected. Place the band selection cursor (small black arrow) on a band
of a reference lane by clicking on it with the mouse and then clicking on its corresponding global
standard lane band marker (the selected band marker will turn white). You can also drag the
mouse within the gel strip (hold down the <Tab> key to take off “Snap to Peaks” and move pixel
by pixel).
Note: Make sure to click the band first and then the global standard band marker to ensure the
correct band marker is highlighted. Enlarging the image using the zoom buttons helps with
placement of band markings.
Once both of the markers are correctly placed, press the <Enter> key to insert the normalization
marker. If you make a mistake in placing the normalization marker, then you can press the
<Delete> key to erase it. Repeat these steps to mark the rest of the bands in the reference lanes.
Another option is to mark only some of the bands for each reference lane. Click on the “Auto
assign reference positions” button to have BioNumerics search and place the normalization
markers. When the “Confirmation” window appears stating, “Do you want to preserve the existing
bands?” click “Yes” to save the selected bands that have been marked. Double-check to make sure
the correct bands in the reference lanes of the gel were matched up with the correct band markers.
After the bands for the reference lanes have been marked, and their placements are correct, then
click on the “Show normalized view” button to show the gel strips as they will appear when
normalized. If changes are made in the normalized view, click on the “Update normalization”
button to update normalization.
To check normalization, click on “Normalization” and “Show Distortion Bars” (in version 3.5,
this option is saved). The gel strip lanes will now show colored distortion bars to the right of the
gel lanes. The colors in the distortion bars indicate the degree of adjustment that was made to each
section of the gel lane. The colors run from light blue for a small adjustment to red and black for a
large adjustment. An unusually high amount of orange, red or black in the distortion bar may
indicate a normalization error, and should be examined. The colors that are seen should be
consistent as they move horizontally across the gel lanes. In the case of an abnormal gel run, dark
distortion bars may remain, even if normalization was performed correctly.
Once the normalized gel strips have been checked for any normalization errors, click on the “Next
step” button to proceed to Step 4 to complete band marking.
Click on the “Auto search bands” button from the toolbar to have BioNumerics search the lanes
for band positions. When the “Band search” window appears, accept the default search values and
click “OK” to perform the band auto search. If the gel is atypical, adjust the minimum profiling
percentage. When the “Confirmation” window appears with a question that asks, “There are
already some bands defined on the gel. Do you want to keep the existing bands?” click “Yes” to
preserve the existing bands marked, “No” for BioNumerics to auto search for all bands again, or
“Cancel.”
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After bands are found by the auto search function, look at the whole gel and delete erroneous or
unnecessary bands. Be sure to go through each lane individually and check band markings by
looking at a printout of the gel image. This will ensure that extra bands, such as those near the top
or bottom of a TIFF image, are deleted. If artifacts are present on a TIFF image or the TIFF image
is dark, the auto search function may find many extra bands.
Refer to the document “PulseNet Gel Analysis Guidelines” (PND04) for more information on how
to mark bands.
To manually insert a band mark, click on the position where you would like the band, and this will
put the band placement cursor where the band mark will be placed. Press the <Enter> key to insert
the band mark. To remove a band mark, highlight the band mark and press the <Delete> key. If
you are uncertain about a band mark, then you can classify a band mark as uncertain (press <F5>),
and this mark will not be included in comparisons (dendrograms, etc.) unless you choose to have
them included. Do not place more than two uncertain bands per gel. If you need more, you should
rerun the corresponding isolates or the entire gel. To access bands that may be marked outside of
the standard, you can un-normalize the gel.
Once all of the lanes have been checked, click the “Save” button to save the normalized gel strips
with band marks. Close the Fingerprint data window after saving. This completes the analysis.
5.8 Changing enzymes/Fingerprint type
5.8.1
It is now time to link lanes; but first, make sure the correct fingerprint type (e.g., PFGE-XbaI,
PFGE-BlnI, etc.) is indicated. If the lanes are linked with incorrect fingerprint types, possible
duplicate entries in the database can occur.
5.8.2
To change the fingerprint type, right-click on the desired lane and select “Change fingerprint type
of lane,” then select the appropriate enzyme.
5.9 Linking isolates to patterns/database entries
5.9.1
To link a lane, either right-click on the desired lane and select the “Link lane” option or highlight
the lane and click on the pink arrow on the top toolbar.
5.9.2
The “Link lane” window will appear and is where a unique isolate number or identifier should be
entered. This code will be known as a “Key” in BioNumerics. Click “OK” after entering the new
key. If the key is not currently present in the database, then a “Confirmation” window appears to
ask if you would like to create the new key. If the key is correct, then click “Yes.” If the key is
incorrect, click “Cancel” and reenter the key to make sure the key is correct.
5.9.3
If an isolate is a re-run, a different “Confirmation” window appears to ask if you would like to
create a duplicate key. If you would, click “Yes.” This will now create the isolate with a “/#1” at
the end; all the demographic information will stay the same.
5.9.4
Do not upload isolates with a “/#” at the end of them. Pull up all versions of the pattern and choose
which one looks the best and should be included in the national database. Double-click on the
isolate and click on the “Edit Database Fields” icon (lightning bolt with green “i”). In the
“Patterns” box, click on the button that corresponds to the “best pattern.” In the PFGE fingerprint
box that opens, click on the “Make first pattern…” button (this button will not appear if the pattern
you have chosen is already the first pattern). If the duplicate that was just linked is the best pattern,
it will no longer have a “/#” behind it and can be uploaded.
5.9.5
Do not link or upload standard/reference lanes. The only exception to this rule is for submission of
certification results.
5.9.6
To enter isolate information for a gel lane, double-click on the desired lane in the “Fingerprint
file” window. This will open the “Entry edit” window. Click on the “Edit database fields” (green
“i”) button to open the customized PulseNet “Entry properties” window. Note that data can be
entered in the “Entry edit” window; however, the data will be lost if you then open the “Entry
Properties” window without first clicking “OK.”
5.9.7
Enter the isolate information into data fields in the customized PulseNet “Entry properties”
window. Most of the data fields use drop-down menus (pick lists) containing fixed values, as well
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STANDARD OPERATING PROCEDURE FOR TIFF IMAGE ANALYSIS
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as previous entries that were used, however, data can also be typed into these fields. In order to
enter correct serotype, use only the drop-down menu to enter serotype or antigenic formula
information for any specific isolate.
Antibiogram types can be entered for an isolate by clicking on the “Antibiotics” button to open the
isolate antibiotic screen. Set an antibiotic resistance level using the pick lists. To add an antibiotic
to the list click “Add,” and enter the new antibiotic name into the text box and click “OK.”
However, only the antimicrobials on the original list will be uploaded. Click “OK” when done.
Repeat entering information for all isolates on the gel.
5.10 Uploading data
5.10.1 Connecting to the PulseNet server is a two-part process. First, authenticate to the CDC firewall
using a SecurID key fob issued by PulseNet. You must pass analysis certification for each
organism database in order to have access to each database. If you are certified for analysis, but do
not have a SecurID, contact the PulseNet Database Team at the CDC. Next, connect to the server
using the BioNumerics software.
5.10.2 After connecting to the server, open the gel with the isolates that need to be uploaded. Click
“Submit fingerprint file to server” (lightning bolt with a blue arrow). In the “Submit fingerprint”
window, select only the lanes that need to be uploaded. Check or uncheck “Submit antibiotics
data” and “Submit biochemical data” as necessary. Then click “OK” to upload the isolates to the
PulseNet database.
5.10.3 If you need to resubmit gel information (i.e., you need to update a serotype or demographic
information), make sure to select all lanes that were previously submitted to the database.
Otherwise, those lanes not selected will become unlinked in the national database.
5.11 Creating comparisons and performing cluster analyses
5.11.1 Select the isolates that you would like to include in your comparison:
5.11.1.1 <Ctrl> + left click; <Shift> + left click; or left click + <space bar>
5.11.2 Or you can select isolates by querying the local database:
5.11.2.1 Several queries can be performed using the script icons on the main
BioNumerics screen.
5.11.2.2 There are queries for: isolate information, pattern information, antibiotics
resistance, and biochemical information.
5.11.2.3 The binoculars can be used to search for information in any field.
5.11.2.4 Use a wildcard (*) around the search criteria to ensure that all entries with the
search string are found.
5.11.3 In the upper toolbar click “Comparison” and select “Create new comparison.”
5.11.4 When the comparison screen opens, highlight the enzyme you wish to show by clicking on the
picture of the gel located next to the enzyme in the bottom toolbar.
5.11.5 In the upper toolbar click on “Clustering” and select “Calculate,” then choose “Cluster analysis
(similarity matrix)…”
5.11.6 In the Comparison settings window the Similarity coefficient should be Band based using the Dice
coefficient, and the Dendrogram type should be set to UPGMA.
5.11.7 You may have to adjust the Position tolerance settings for your dendrogram. You can do so by
clicking on the “Position tolerances…” button; however, PulseNet suggests using an Optimization
of 1.50 and a Position tolerance of 1.50. Click “OK” to close the Position tolerance settings
window.
5.11.8 Click “OK” in the Comparison settings window to create the dendrogram tree structure in the
dendrogram area of the Comparison window.
5.11.9 To compare two isolates, select the entries and click “Comparison” in the upper toolbar. Select
“Compare two entries” and highlight the enzyme that you wish to show.
6. FLOW CHART:
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7. BIBLIOGRAPHY:
Weinberg, Sandy. GOOD LABORATORY PRACTICE REGULATIONS. Second edition. Marcel Dekker, Inc.
USA (1995).
8. CONTACTS:
8.1 PulseNet Database Team
(404) 639-4558
PFGE@cdc.gov
9. AMENDMENTS:
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STANDARD OPERATING PROCEDURE FOR ACCESSING AND USING THE
PULSENET WORKSPACE ON CDC TEAM
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Effective Date:
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08
1.
PURPOSE: To explain the procedure for obtaining access to and using the PulseNet Workspace
on CDC Team.
2.
SCOPE: This procedure applies to all persons who need or have access to the PulseNet Workspace
on CDC Team.
3.
DEFINITIONS/TERMS:
3.1 PulseNet Workspace on CDC Team: A closed, web-based discussion forum used for
communication among PulseNet participants. The PulseNet Workspace on CDC Team is
open to all laboratory staff at PulseNet participating laboratories. Epidemiologists working in
collaboration with these laboratories, when approved by the PulseNet laboratory contact, U.S.
food regulatory staff, and PulseNet International representatives may also have access. The
PulseNet Workspace on CDC Team is not open to university or private industry personnel
unless their inclusion is deemed to be in the interest of public health. Throughout this
document, the PulseNet Workspace on CDC Team will be referred to as “CDC Team.”
(http://team.cdc.gov).
3.2 PFGE: Pulsed-field Gel Electrophoresis
3.3 CDC: Centers for Disease Control and Prevention
3.4 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths,
Belgium
3.5 TIFF: Tagged Image File Format, a file of a gel image that can be analyzed in BioNumerics
3.6 EIS: Epidemic Intelligence Service. EIS Officers are epidemiologists who are given two-year
appointments to help in the investigation of outbreaks.
3.7 PulseNet Liaison: Contact for Enteric Diseases Epidemiology Branch epidemiologists and the
PulseNet Database Team.
3.8 PFGE Inbox: An e-mail account that is maintained and checked by all database managers at
CDC. The address is: PFGE@cdc.gov.
3.9 EDLB: Enteric Diseases Laboratory Branch
3.10 Area Laboratory: Laboratory, designated by CDC, which has agreed to assume responsibility
for additional PulseNet duties for laboratories within their support zone. The current Area
Laboratories include CDC, Massachusetts, Michigan, Minnesota, Texas, Utah, Virginia, and
Washington.
3.11 Cluster: A group of isolates, identified within the past 30 days for Yersinia; 60 days for
Salmonella, E. coli, Shigella, Campylobacter, and Vibrio; and 120 days for Listeria, with the
same serotype with indistinguishable PFGE DNA patterns by one or more restriction
enzymes
3.12 STEC: Shiga toxin-producing E.coli. STECs are the most virulent of the diarrheagenic
pathogens under surveillance in PulseNet.
3.13 NDA: A Non-disclosure Agreement is a legal contract between at least two parties that
outlines confidential materials or knowledge the parties wish to share with on another for
certain purposes, but wish to restrict from generalized use. In other words, it is a contract
through which the parties agree not to disclose information covered by the agreement. An
NDA creates a confidential relationship between the parties to protect any type of trade
secret. As such, an NDA can protect non-public business information.
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Effective Date:
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PROCEDURE:
4.1
CDC Team Access
4.1.1 All personnel who wish to access CDC Team must send an email to the PFGE Inbox
(PFGE@cdc.gov) with “CDC Team Request” in the subject line.
4.1.1.1 The requestor should include the person’s name, email address, organization
and phone number in the email.
4.1.1.2 By the rules set up in the PFGE inbox, the request email will automatically be
filtered to the “CDC Team Requests” folder
4.1.2 State and local epidemiologists must request access to CDC Team through their
PulseNet laboratorian. The laboratorian will then follow the steps mentioned in 4.1.1
to request access for their epidemiologist(s).
4.1.3 The CDC PulseNet liaison(s) must request access for EIS officers by the steps
mentioned in 4.1.1 above.
4.1.4 The PulseNet Database Unit Chief determines if access is approved or disapproved.
4.1.5 If access is disapproved, the PulseNet Database Unit Chief notifies the requestor
immediately.
4.1.6 If access is approved, the PulseNet Database Unit Chief forwards the requestor’s
email to PulseNet Computer Support.
4.1.7 PulseNet Computer Support creates a login and password, which allows access to the
PulseNet Non-disclosure Agreement (NDA) on the PulseNet Workspace on CDC
Team and notifies the subscriber via email. The new subscriber must print, read, sign
and return to PulseNet as described in Appendix PND03-1. Signing the nondisclosure agreement indicates that the user will log into CDC Team, change their
password, and agree to follow the CDC Team policy on sharing information (section
4.2 below).
4.1.8 When CDC receives the faxed copy of the completed Nondisclosure Agreement,
EDLB Administrative Support will submit an ITSO service request to grant access to
the PulseNet forums. EDLB Administrative Support will also receive the mailed
copy of the Nondisclosure Agreement and file with the faxed copy and add received
dates for both documents in the subscriber database.
4.1.9 PulseNet Computer Support receives the ITSO service request, adds subscriber to the
subscriber database, gives access to PulseNet forums on the PulseNet Workspace on
CDC Team and closes the ITSO service request.
4.1.10 Passwords must not be shared or disclosed to anyone, for security purposes.
4.1.11 When a user leaves their current position or is no longer performing PulseNet duties,
they or their supervisor are required to notify CDC so that CDC Team access will be
terminated. CDC may be notified by emailing PFGE@cdc.gov with “CDC Team” in
the subject line or calling (404) 639-4558.
4.1.12 Once CDC is notified of a subscriber’s departure, the PulseNet Database Unit Chief
submits an ITSO service request to have that subscriber removed from CDC Team.
4.1.13 PulseNet Computer Support receives the ITSO service request, removes the
subscriber from the subscriber database and removes their access to the PulseNet
Workspace on CDC Team and closes the ITSO service request.
4.2
CDC Team Policy on Sharing Information
CDC Team postings contain preliminary information on presumptive disease clusters and
ongoing outbreak investigations. Therefore, postings are not appropriate for sharing with
persons outside public health and food regulatory agencies. If you would like to share
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messages with persons not directly associated with public health or food regulatory
agencies, we require that you obtain prior approval from the person or agency that posted
the information. We would appreciate your strict compliance with this policy. Violation
of this policy will result in loss of access to CDC Team.
4.3
CDC Team Administrative Roles
There are four types of CDC Team users, each with a different level of privileges and
security. In descending order, these are the CDC Team Administrator, Manager,
Moderator, and User.
4.3.1 CDC Team Administrators have complete control over the entire CDC Team
website. This level of administration is managed by a contractor to CDC, with
oversite of the CDC Information Technology Services Office.
4.3.2 PulseNet Workspace Managers have control over the forums, moderators, and
users on the PulseNet Workspace. Managers are responsible for adding and
removing forums, setting access rights on forums, and adding and removing
participants to CDC Team. Managers are also responsible for assigning Moderators
to select forums. This level of administration is managed by PulseNet Computer
Support. The PulseNet Database Unit Chief has backup responsibilities at this level.
4.3.3 PulseNet Workspace Moderators have full responsibility for maintaining assigned
forums by responding to, updating, archiving, and removing information as described
in 5.2. Moderators can edit or delete postings, retrieve them from the archives, and
modify forum settings. This level of administration is managed by the PulseNet
Database Team.
4.3.4 PulseNet Workspace Users can read, post, and respond to messages on CDC Team.
Users can delete messages that they post. Users do not necessarily have access to all
forums on CDC Team.
4.4
CDC Team Setup
4.4.1 CDC Team is divided into several organized forums. Topics should be posted within
the appropriate forum. Topics may be added or removed at the discretion of the
Administrator and Manager.
4.4.2 Clicking on a forum allows the user to see the topics and threads posted within that
forum.
5. RESPONSIBILITIES:
5.1 Posting Cluster or Outbreak-Related Topics and Responses on CDC Team
5.1.1 Cluster and outbreak information should be posted on CDC Team for quicker and
more efficient reporting.
5.1.1.1 Suggestions for when to post a cluster
5.1.1.1.1 If you have a cluster of two or more indistinguishable patterns that have
an epidemiological connection you should post
5.1.1.1.2 If you have a cluster of three or more isolates (same serotype) with a
rarely seen pattern in your laboratory you should post
5.1.1.1.3 If you have a cluster of a common pattern, then monitor that cluster to
see if there is a significant increase in your laboratory before posting
5.1.1.1.4 Pattern and serotype commonality differ within each community,
therefore the decision to post a cluster is up to each individual laboratory.
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5.1.1.1.5
If you have questions about when to post, you can always contact CDC
or your PulseNet Area Laboratory
5.1.1.2 Isolate patterns must be uploaded to the PulseNet national databases before
posting to CDC Team (if not certified, the patterns must be submitted via email
to the PFGE inbox).
5.1.1.3 The following information must be included in the CDC Team posting and/or
sent by email to the PFGE Inbox (see Appendices PND03-2 and PND03-3 for
example postings):
5.1.1.3.1 For initial postings, a PulseNet bundle file and/or a TIFF containing the
cluster or outbreak patterns should be provided
5.1.1.3.1.1 If a PulseNet bundle file is provided, this will allow users to
compare the isolates in BioNumerics without having to reanalyze
the gel image. Therefore it is recommended that a PulseNet
bundle file be posted. NOTE: If a PulseNet bundle file is not
used, it will be deleted from CDC Team by the forum moderator
and the poster will be asked to post a PulseNet bundle file.
5.1.1.3.1.2 If only a TIFF is posted, isolate information, including but not
limited to the items listed in sections 5.1.1.3.2 - 14, must be
provided in the posting
5.1.1.3.2 Name of the submitting laboratory
5.1.1.3.3 Which enzyme(s) used—state if second enzyme is pending
5.1.1.3.3.1 Second enzyme should always be run for E. coli O157:H7 and
other STECs and Listeria monocytogenes per the “Second
enzyme recommendations” document posted on CDC Team.
5.1.1.3.4
5.1.1.3.5
5.1.1.3.6
Serotype, if applicable
Shiga toxin type for E. coli O157 and other STECs if available
Lanes that contain isolates that are part of the cluster or outbreak (if TIFF
is posted)
5.1.1.3.7 Collection and/or isolation date and date received when available
5.1.1.3.8 Information on additional isolates that may be part of the cluster or
outbreak
5.1.1.3.9 Additional information about the cluster pattern, such as number of
isolates and frequency of pattern in your local database
5.1.1.3.10 In an original posting, CDC pattern designations should not be posted by
anyone other than CDC Database Team members, even if the pattern is
listed as confirmed within the national database.
5.1.1.3.11 Postings should only include information pertaining to the laboratory’s
local data (any observances or questions regarding the national database
should be limited to emails directly to the PulseNet Database Team,
PFGE@cdc.gov)
5.1.1.3.12 Any available epidemiological information
5.1.1.3.13 Personal identifier information such as patient names should not be
posted to CDC Team
5.1.1.3.14 Company names should not be posted unless it is part of official
published media (i.e. press release, ProMed, newspaper article, Epi-X,
etc.)
5.1.1.4 For laboratory response postings, the following information must be provided:
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5.1.1.4.1
If a laboratory has seen the cluster pattern within the past 30 days for Y.
pestis, 60 days for E. coli, Shigella, Salmonella, Campylobacter, and
Vibrio or 120 days for Listeria, then an isolate number or file name
should be provided in the posting.
5.1.1.4.2 It is not necessary for a lab to post indistinguishable isolates that are
already included in a CDC cluster/outbreak summary spreadsheet on
CDC Team. Please continue to post new isolates that have not been
included in these spreadsheets.
5.1.1.4.3 It is mandatory that all patterns reported as indistinguishable be uploaded
to the PulseNet national databases or emailed to PFGE@cdc.gov.
5.1.1.5 Epidemiologist information
5.1.1.5.1 Once a PulseNet database manager from CDC becomes aware of which
CDC epidemiologist is following a particular cluster or outbreak, that
person’s name and email address will be posted at the top of the initial
posting for that cluster/outbreak.
5.1.1.5.1.1 It should be noted that public health laboratorians should go
through their epidemiologists when contacting the CDC
epidemiologists regarding an investigation.
5.1.1.5.2 For epidemiologist response postings
5.1.1.5.2.1 Epidemiologists are encouraged to post epidemiological
information on CDC Team. We encourage laboratorians and
epidemiologists to work together before posting information on
CDC Team.
5.1.1.5.2.2 For ease of finding these postings, please put “STATE EPI
UPDATE” in bold, blue letters at the top of the posting
5.1.1.5.2.3 Personal identifier information such as patient or restaurant
names should not be posted to CDC Team (see 5.1.1.3.13 and
5.1.1.3.14 above)
5.2 CDC Team Management
5.2.1 Topic Removal
5.2.1.1 Once a topic has been posted to CDC Team, a Forum Moderator should
respond within two business days of the original posting.
5.2.1.2 Moderator response postings for outbreaks or clusters should include (but are
not limited to) an outbreak/cluster code, pattern number assignments,
frequencies of patterns, any pertinent epidemiological data known, and a
PulseNet bundle file containing the patterns and isolate information (if one has
not already been posted).
5.2.1.3 Topics should be renamed with the following format: <Outbreak Code>
<(pattern number)>_<LabID of posting lab>_<Organism and/or Serotype>.
5.2.1.4 Updated information should be posted by the forum Moderator as necessary. A
summary will be posted when the cluster or outbreak comes to an end.
5.2.1.5 After three weeks of inactivity, the original posting and all response postings
will be summarized (if not already) and archived according to the “PulseNet
CDC Team Guidelines for Archiving Postings” (see Appendix PND03-4). The
summary should include a “cut-thread” which states that if there are no further
responses within the next seven days, the forum Moderator will remove this
topic from CDC Team. After seven days, if there have been no responses, the
topic will be archived.
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6. FLOW CHART:
7. BIBLIOGRAPHY:
8. CONTACTS:
9. AMENDMENTS:
9.1 This SOP replaces the former SOP PND03 PulseNet Listserv Access and Use describing
the use of WebBoard as the PulseNet communication listserv. As of February 29, 2008 the
contents of WebBoard were moved to the PulseNet workspace on CDC Team. CDC Team
is now being used as the PulseNet web-based discussion forum.
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Appendix PND03-1
PulseNet CDC Team Non-disclosure Agreement
The following information is posted in the PulseNet Workspace on CDC Team.
Welcome to PulseNet – CDC Team!
In order to comply with Health and Human Services security requirements, we are required to obtain a
signed non-disclosure agreement from each PulseNet – CDC Team subscriber.
The following is the PulseNet – CDC Team Policy on Sharing Information. It is displayed on the
PulseNet – CDC Team as a reminder:
PulseNet – CDC Team Policy on Sharing Information
Often PulseNet – CDC Team postings contain preliminary information on presumptive disease
clusters and ongoing outbreak investigations. Therefore, PulseNet – CDC Team postings are not
appropriate for sharing with persons outside public health and food regulatory agencies. If you
would like to share PulseNet – CDC Team messages with persons not directly associated with
public health or food regulatory agencies, we require that you obtain prior approval from the
person or agency that posted the information.
We would appreciate your strict compliance with this policy. Violation of this policy will result in
loss of access to the PulseNet – CDC Team.
Thank you for your cooperation
Peter Gerner-Smidt MD, PhD
Centers for Disease Control and Prevention
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PRINT, MAIL AND FAX THIS PAGE ONLY
PulseNet – CDC Team Non-Disclosure Agreement
By signing this non-disclosure agreement, you agree that you will not share information from the
PulseNet – CDC Team with personnel not associated with the public health community, and that you will
change your password the first time you logon to the PulseNet – CDC Team.
I have read the PulseNet – CDC Team Policy on Sharing Information and agree not to share
information from the PulseNet – CDC Team with personnel not associated with the public health
community.
I will change my password when I log onto the PulseNet – CDC Team for the fist time.
Name:
Laboratory/PulseNet Affiliation:
Email Address:
Signature:
Date:
Please print this page, then fax and mail to:
Michael Korth
CDC
NCZVED DFBMD EDLB
1600 Clifton Road, NE
Bldg 1 Room 341B
Mailstop C03
Atlanta, GA 30333
Fax: (404) 639-3333
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Appendix PND03-2
Example: Correct PulseNet CDC Team Initial Posting
Topic:
Conf:
From:
Date:
E. coli O157:H7 in GA
E. coli
Jane Doe JDoe@emailaddress.gov
Monday, January 16, 2008
GA is experiencing a cluster (5 cases and 1 food) of E. coli O157:H7 in the Atlanta area. The following
information pertains to the attached PulseNet bundle file, GA080374PN. The associated TIFF was
uploaded to the PulseNet national database for E. coli this morning. Both enzymes have been run for
these isolates.
Isolate #
GA1000
GA1001
GA1002
GA1003
GA1004
GA1005
Collection
date
01-05-08
01-04-08
01-08-08
01-08-08
01-06-08
01-03-08
Date received
01-08-08
01-07-08
01-11-08
01-10-08
01-08-08
01-06-08
Source Type
Source Site
Human
Human
Human
Food
Human
Human
Stool
Stool
Blood
Hamburger
Stool
Blood
[Patient demographics, including age, sex, and county, is not necessary on CDC Team posting, but needs
to be included in the email to PFGE@cdc.gov.]
Within our local database, this pattern is not common; we have seen it only once before. The isolate
number is GA0076, and it was uploaded in January of 2008. As of this date, we have no epi information,
but the cluster is potentially linked to hamburger.
***GA08374PN.BDL*** (this is the attached PulseNet bundle file)
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Appendix PND03-3
Example: Correct PulseNet CDC Team Response Posting
If lab has seen posted pattern:
Topic:
Conf:
From:
Date:
E. coli O157:H7 in GA
E. coli
Jon Doe Jondoe@email.edu
Monday, January 16, 2008
AL has two possible matches to the GA patterns. These isolates are AL1234 with an upload date of 1-1208 and AL1245 with an upload date of 1-13-08.
***AL08056PN.bdl*** (this is the attached PulseNet bundle file)
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Appendix PND03-4
PulseNet CDC Team Guidelines for Archiving Postings
1. Depending on the pathogen/database, CDC Team postings are archived 3 weeks from the LAST
posting of the topic or thread.
2. Before posting the cut thread, summarize the information from the posting. (i.e., if it was an outbreak,
give pattern numbers, numbers seen from which state(s), commonality of the pattern in the database,
if the outbreak was ever linked back to a food item and/or resulted in a recall.) A template can be
found here: \\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\CDC
Team\admin\Templates\Summary_posting.doc
3. Post a cut thread (see below or go to \\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\CDC
Team\admin\ cut thread.doc) to that posting:
If there are any additional replies or inquiries regarding this topic, [title topic], please be sure they are
submitted as soon as possible. If no further postings are received, the Database Team will remove this
topic from CDC Team in approximately 7 days.
Comments or questions specifically related to this thread may be directed to either [forum manager]
by sending an email to [forum manager’s email] or [Person posting topic], the originator of this thread
by emailing to [originator’s e-mail].
If you need information relating to a conference thread that has been removed from PulseNet CDC
Team, please send an email to PFGE@CDC.GOV.
Thank you,
[Your name]
4. After 7 days, if there have been no responses, the topic can be archived.
5. To archive a posted thread:
• Highlight the entire topic discussion—make sure to include all postings—and copy
• Go to Edit>Paste Special, and choose “Unformatted Text”, and paste the copied topic into the template
document under \\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\CDC Team\admin\templates\
archive_template.doc (it’s about 5 times smaller file size)
• In the Responses to box, type the topic name, and the date of the first posting
• Save the word document under: \\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\CDC
Team\Forum\topic folder\year
• Use the following format for the filename: for outbreaks use: Topic name (ex: 0707COEXK1_(EXKX01.0001)_CO_E. coli O121/O26) or for non-outbreaks use: YYYY-MM-DD_LabIDTopic (ex: 2002-09-17_CDC-Serotyping and Ribotyping Outbreak.doc); the date is the date of the
first posting
• Save any attachments that are NOT TIFF or bundle files into an appropriate “attachments” folder.
6. Once the thread is saved, the thread can be deleted from CDC Team.
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7. Open the report document located under: \\Cdc\project\CCID_NCZVED_DFBMD_PulseNet\CDC
Team\Reports\year\WebBoard_YYYY.xls (choose the appropriate year).
• Under the appropriate tab, enter the following information: Sender, LabID, Date Sent, Date Ended,
Forum, Topic, Number of Responses, and Duration on CDC Team (this can be calculated in Excel by
subtracting ‘Date Sent’ from ‘Date Ended’
Things to consider:
1. Frequency of sending threads may depend on the outbreak and/or organism (epidemiology). For large
multi-state outbreaks, the posted thread may consist of postings over a month long; however, small
isolated clusters may only last for a couple of weeks. For Shigella, since the transmission is personto-person (PTP) then posted threads may consist of postings over a period of 2-3 months. There needs
to be an established guideline for sending of these types of posted threads.
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CODE: PND04
STANDARD OPERATING PROCEDURE FOR PFGE GEL ANALYSIS
Effective Date:
11 16 2009
1.
PURPOSE: To describe the guidelines for standardization of TIFF image analysis in order to accurately
compare PFGE patterns between PulseNet participating laboratories.
2.
SCOPE: This procedure applies to all analysis submitted to PulseNet, thereby allowing comparisons of
results with other PulseNet laboratories.
3.
DEFINITIONS/TERMS:
3.1 PFGE: Pulsed field Gel Electrophoresis
3.2 TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics
3.3 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
4.
BACKGROUND: PulseNet Gel Analysis Guidelines
Computerized gel analysis with BioNumerics facilitates comparison of PFGE patterns from isolates of foodborne
bacteria on gels produced by all certified PulseNet laboratories. It is critical that excellent quality gels that have been
analyzed accurately are submitted to the PulseNet on-line databases for valid comparison of results.
Computer analyses (from lane definition to normalization to band finding) should always be checked against a copy
of the actual gel image being analyzed, as visual (i.e., manual) analysis is the gold standard. Analysis is only as good
as the TIFF of the gel image being analyzed. For this reason, each gel image should be of the highest quality
possible. Consistency from run to run is critical. Factors such as fuzzy bands, smearing, high background, the
presence of shadow bands, and running the gel too short or too long can make TIFFs of the images needlessly
difficult to analyze and can compromise comparisons to patterns in the national databases. Gels that do not yield
TIFFs with acceptable quality should be rerun.
Sometimes, suggestions to improve gel quality come about after computer analysis of what appears to be a good gel
visually. Factors such as consistent slants of lanes and bands, band distortions, light bands at the bottom of the gel or
in a few lanes, extreme differences in the band spacing of the standards compared to the global standard, debris or
artifacts that affect analysis, and poor band resolution can be more apparent during computer analysis of a TIFF than
upon visual inspection. When factors such as these are noted during analysis, the person performing the analysis
should suggest and/or implement changes in gel preparation so that gel quality will be of the highest quality
possible.
5.
RESPONSIBILITIES/PROCEDURE:
5.1 Standards
5.1.1 Standards are very important for normalization. Standards can also be used to gauge consistency from
gel to gel and provide clues about how to evaluate the test isolates. Evaluation of the standards may
include the following:
5.1.1.1 Are the standard bands consistently the same thickness or intensity from gel to gel?
5.1.1.2 During normalization, note how the standard pattern on the gel matches the band pattern of the
reference standard. If BioNumerics must stretch or shrink the pattern to match the reference
standard, is the stretching or shrinking the same from gel to gel? If a sudden change occurs, an
error might have occurred during the run such as the wrong electrophoresis program used to run
the gel. Using the show distortion bars feature will aid in visualizing these types of problems and
assist with correct normalization.
5.1.1.3 From gel to gel, is the standard pattern consistently the same or does the gel resolution vary
enough to affect the standard pattern (i.e., doublets appear sometimes and single bands appear at
other times in certain areas of the pattern)? For normalization purposes on E. coli O157:H7 gels
(for example), 15 normalization markers on 15 single bands can be placed on the Salmonella
Braenderup H9812 standard. However, some laboratories are resolving doublets where single
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bands appear on the global standard near the bottom of the H9812 pattern. If doublets resolve,
mark the normalization bands carefully (see IB. below).
5.1.2 Not all observed bands in a standard lane are used to normalize. If an area of a standard lane resolves as a
doublet on a test gel but appears as a single band on the global standard, a single normalization marker
(upper band, lower band or between the two bands) should be marked in this area on the test gel.
However, for band finding (step 4), a doublet should be marked on the test gel. Normalization (step 3)
will not be affected if areas of the standard lanes are normalized correctly as single bands but marked as
doublets during band finding.
5.1.3 If shadow bands appear in the standard lanes, shadow bands could be present in test lanes. Ideally, the gel
should be repeated. Lanes containing shadow bands are difficult to analyze. Shadow bands probably
indicate incomplete restriction and should not be present on a gel. Repeat the gel performing restriction
with more units of enzyme, for a longer amount of time and/or with a different lot of enzyme. Wash the
plugs at least two more times with TE Buffer before restriction is repeated.
5.2 Before analysis
5.2.1 Consistently taking pictures where the gel image fills almost the entire window on the imaging equipment
screen (without cutting off wells or the bottom of the gel) should improve consistency in normalization
and band finding.
5.2.2 Print out or open up on a computer screen the gel image to be analyzed. Always analyze while looking at
a copy of the actual gel image (either on-screen or hard copy). The copy of the gel image should steer
decisions on band finding, such as deciding if an area is a doublet or a single band.
5.2.3 If shadow bands are present or if incomplete restriction is suspected, rerun the sample(s) or, if necessary,
the entire gel. Lanes with incomplete restriction of DNA are difficult to analyze and may yield a different
pattern when the PFGE is repeated. Results from isolate patterns showing incomplete restriction are not
always reproducible and cannot be accurately compared with patterns from other isolates in a local
database or with the national database.
5.3 BioNumerics – Step 1 Strips
5.3.1 At the beginning of the analysis, assign the TIFF to the enzyme used on the gel. Two enzymes are used,
lanes restricted with secondary enzymes should be changed after analysis but before lane linkage to the
local database (see section 5. 7of this document).
5.3.2 To define the gel strips, place the sides of the green box frame on the edges of the first and last lanes of
the gel. Place the top of the green box frame directly under the wells. Place the bottom of the green box
frame approximately 1-2 cm below the lowest band of the Salmonella Braenderup H9812 standard on a
full-size image. (On a gel following the PulseNet standardized procedure where the last band of the
standard is 1-1.5 cm from the bottom of the gel and the gel image fills the entire window on the imaging
equipment screen, 1-2 cm below the last band of the standard should be at the bottom of the gel.) This
will standardize lane lengths, which will produce more accurate normalizations and improve comparisons
with the national database.
5.3.3 After the lanes are defined, adjust the “thickness” under “edit settings” to increase or decrease the
thickness of the defined gel strips so that the left and right edges of the strips are just outside the outer
edge of the bands. This will ensure that the bands will appear in a size for proper analysis. If the gel strips
are defined incorrectly (too wide), the resulting narrow bands with large white patches on either side of
the bands are difficult to analyze. Gel strips may need adjustment when switching between 10-well and
15-well gels and also if the gel image is captured differently (i.e., close up versus farther away) from gel
to gel. The width of an individual lane can be adjusted independently, if necessary.
5.3.4 Using the “linear adjustment” in the “edit tone curve” feature may provide better band clarity for
analysis.
5.3.5 In the edit tone curve feature, enhancing weak bands is sometimes useful to detect bands accurately.
However, when using the edit tone curve feature to enhance weak bands (primarily seen at the bottom of
the gel), too much enhancement of weak bands may make the stronger stained bands in the top and
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middle portions of the pattern appear too thick and make them difficult to analyze. Always compare band
markings in the software to a copy of the original gel image.
5.3.6 Increasing contrast is sometimes useful to detect bands accurately.
5.3.7 Do not use background reduction or spot removal on gel images submitted to CDC. The different usage
of these BioNumerics tools among PulseNet participating laboratories causes problems when performing
comparisons within the national databases. Participating laboratories may use background reduction and
spot removal in their local databases, but should remove these features before submitting analyses to the
national databases. CDC does not recommend the use of spot removal.
5.4 BioNumerics - Step 2 Curves
5.4.1 If bands in the test lanes or the standard lanes are distorted or contain artifacts and the gel image must be
analyzed without rerunning the gel, ensure that the curve strips are placed accurately.
5.5 BioNumerics - Step 3 Normalization
5.5.1 If artifacts (e.g., specks or dots) appear near a band on the TIFF image, ensure a normalization marker is
not placed on the artifact instead of where the actual band is on the TIFF image.
5.5.2 Check normalization using the “distortion bars” function (under the Normalization menu). The color of
the distortion bars should be the same moving across the gel. If a high amount of orange or red distortion
bars or distortion bar colors that change as they move horizontally across the gel strips appear,
normalization marker placement should be examined. If dark distortion bars are present and uneven
across the gel and the normalization marker placement is correct, the gel should be rerun, verifying that
the correct running conditions are used.
5.5.3 In BioNumerics, when Step 3 (Normalization) is finished, take off the normalized view before moving to
Step 4 (Bands). If the image is left in the normalized view for band finding, bands could appear below
the lanes which cannot be deleted when you move to Step 4 (Bands).
5.6 BioNumerics - Step 4 Bands
5.6.1 After bands are found by the auto search function, look at the whole gel and delete obvious extra bands
first before carefully going through each lane and matching band markings to a copy of the gel image.
This will ensure that extra bands, such as those near the top or bottom of a TIFF image, are deleted. If
artifacts are present on a TIFF image or the TIFF image is dark, the auto search function may find many
extra bands.
5.6.2 Review the bands detected by the auto search function.
5.6.2.1 Mark bands carefully. Use the peak of the shoulder on the densitometric curve if band
placement is in question. If a band is considered a singlet, ensure that the single band is placed
correctly. Bands should be marked in the middle of areas considered single, solid bands.
5.6.2.2 Mark bands as they appear on a copy of the actual gel image. Defer to a hard copy of the image
when determining if a questionable area should be marked as a doublet or a single band.
5.6.2.3 All lanes containing profiles, which by visual inspection are obviously indistinguishable,
should be marked uniformly.
5.6.2.4 If artifacts (e.g., specks or dots) appear near a band on the TIFF image, make sure a band is not
placed on the artifact instead of where the actual band is on the gel image.
5.6.3 When deciding if a band is a doublet or a single band, look for white space in the middle of the area or
indentations (i.e., doughnuts or shoulders) on the sides of the area that separate two bands. If indentations
or white space are apparent on a copy of the TIFF, the area is most likely a doublet. If the indentations or
white space are questionable, the area probably should be marked as a single band. Separate peaks on the
densitometric curve may also provide clues as to the band designation of the area.
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Doublet
Single band
5.6.4 The uncertain band feature of BioNumerics should be used sparingly. If an area on an excellent or good
gel is either a doublet or a single band and the criteria in 5.6.3 does not aid in band marking, the
uncertain band feature may be used. If the gel quality is fair or poor, repeat the gel and produce a good
quality pattern before marking bands.
5.6.5 As overall gel quality improves, resolution will become sharper. If an area that previously resolved as a
thick singlet becomes a doublet or triplet on subsequent gels, that area should be marked exactly as it
appears on each gel.
5.6.6 When using the Salmonella Braenderup H9812 standard, visible bands of test isolates should be marked
down to ~20 kb (the bottom band used for normalization in the H9812 standard).
5.6.7 In almost all cases, band intensity differences are not reliable enough for use in assigning pattern
numbers. Two patterns that are indistinguishable except for one or more band intensity difference(s) will
be assigned the same pattern number. Isolates with only intensity differences should be reported as
indistinguishable by the test(s) run and the PulseNet criteria.
5.6.8 Most TIFFs analyzed for the national databases are produced by a digital camera system (i.e., Gel Doc).
If an analyzed TIFF was produced by a digital camera system but the band finding is performed while
looking at a picture developed from film, make sure that the band resolution on the digital camera image
is equivalent to that of the film picture. This will ensure that band markings are consistent with the TIFF
image being analyzed and are comparable with other patterns in the national databases.
5.7 Lane linkage
5.7.1 Before linking a lane to the local database, make sure that the fingerprint type (e.g., PFGE-XbaI, PFGEBlnI, etc.) is correct. Changing fingerprints is necessary when more than one enzyme is used for
restriction on a particular gel. If the lanes are linked with incorrect fingerprint types, possible duplicate
entries in the database could occur. At the beginning of the analysis, assign the TIFF to they enzyme used
on the gel. Any lanes restricted with secondary should be changed after analysis but before lane linkage
to the local database. To change the fingerprint type, right-click on the desired lane and select “Change
fingerprint type of lane…” After the fingerprint type is changed, you can link the lane to an entry in the
database. Both fingerprint types should be indicated with a green dot next to the one entry in the
BioNumerics database.
5.8 Review of analysis and submission to CDC
5.8.1 PulseNet participating laboratories should review their band finding and normalization before submitting
to the PulseNet national database, PFGE inbox, or the PulseNet Listserv. After band finding and the
linkage of lanes in a TIFF, normalization and band finding can be roughly checked by analyzing a
dendrogram showing gel strips and band markings.
5.8.2 Submit your laboratory’s best possible gel image and analysis of a particular pattern. If one or more lanes
of an otherwise good gel are unsatisfactory, do not upload the unsatisfactory lanes to the national
database. Rerun unsatisfactory isolates on another gel.
5.8.3 Do not use background reduction or spot removal on gel images submitted to CDC. The different usage
of these BioNumerics tools among PulseNet participating laboratories causes problems when performing
comparisons within the national databases. Participating laboratories may use background reduction and
spot removal in their local databases, but should remove these features before submitting analyses to the
national databases. CDC does not recommend the use of spot removal.
5.8.4 Within your laboratory, regularly check band markings of all analysis-certified personnel to ensure
consistency. This should be included as part of your laboratory’s Quality Assurance/Quality Control
program.
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5.8.5 All TIFF and bundle files must be named according to the PulseNet protocols for naming. TIFFs and
bundle files from each PulseNet participating laboratory should have unique names by laboratory and not
by species when they are sent to CDC. For example, the first TIFF and bundle from GA in 2004 should
be named GA04001.tif and GA04001PN.bdl. This could be a gel containing E. coli O157:H7 or another
organism. The second TIFF and bundle from GA in 2004 should be named GA04002.tif and
GA04002PN.bdl. This could be a gel containing Salmonella or another organism.
5.8.6 After installing the December 2003 Master Scripts, create PulseNet bundle files using the bundle file
lightning bolt icon on the left side of the BioNumerics screen. Bundle files created using this icon will
contain only PulseNet fields and “PN” will automatically be added to the file name.
5.9 After submission to CDC
5.9.1.1
All certified PulseNet participating laboratories have the ability to check the on-line databases
to see if previously submitted patterns have been assigned pattern numbers by CDC. These
pattern names can be downloaded to the local database.
5.9.1.1.1 If a pattern number is followed by a “&” or “@,” the number has not been confirmed by
the CDC Database Team. These pattern numbers are to be considered preliminary and
must not be reported.
5.9.1.1.2 If the band marking is different in the local database than it is in the on-line database,
the CDC Database Team has changed the participating laboratory’s original analysis.
The participating laboratory should review the changes made by the Database Team,
and if necessary, contact CDC to find out why the changes were made. The participating
laboratory can then download the changes to their local database.
5.9.1.1.3 If a pattern status has been marked “unsatisfactory,” the submitting laboratory must
rerun the isolate on another gel. Contact the CDC Database Team if there is a question
about the designation.
5.9.2 If resubmission/reupload of results from the same TIFF is necessary (e.g., for Salmonella serotype
information), highlight all isolates on that gel before reuploading, even if only one lane has results that
are being resubmitted. If all lanes are not highlighted, unlinking could occur.
6.
FLOW CHART:
7.
BIBLIOGRAPHY:
8.
CONTACTS:
9.
AMENDMENTS:
9.1. Statements regarding unsatisfactory patterns having “NG” in the pattern names were removed because the
CDC Database Team no longer uses ‘NG” to denote an unsatisfactory pattern. Please see section 5.9.1.2.3.
9.2. Updated section 5.8.5 and 5.8.6 to include a description of the “PN” for PulseNet bundle files
9.3. References to the BioNumerics Training Manual were removed. Current versions of the training
notebooks are posted on the PulseNet Listserv after each CDC BioNumerics Training Course. Please refer
to those presentations for step-by-step instructions on using the tools in BioNumerics.
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1. PURPOSE: To describe the procedure for setup and use of a SecurID Key Fob.
2. SCOPE: This procedure applies to all PulseNet personnel who are analysis certified and receive a
SecurID Key Fob.
3. DEFINITIONS/TERMS:
3.1. SecurID Key Fob: Token that displays a six-digit passcode. When used in combination with a
four-digit pin number, allows access through the CDC firewall.
4. RESPONSIBILITIES/PROCEDURE:
4.1 Precautions for Protecting Your SecurID Key Fob. For your own protection and that of the
system, always take the following precautions:
4.1.1 Never reveal your PIN or user password to anyone. Do not write them down.
4.1.2 If you think someone has learned your PIN, notify CDC PulseNet to have the PIN cleared
immediately. At your next login you will have to create a new PIN to use.
4.1.3 Exercise care not to lose your SecurID Key Fob or to allow it to be stolen. If your key fob
is missing, notify CDC PulseNet immediately. They will contact the administrator, who
will disable the key fob so that it is useless to unauthorized users.
4.1.4 Do not let anyone access the system under your identity (that is, log in with your PIN and a
code from your SecurID token).
4.1.5 It is essential to site security that you follow your system’s standard logoff procedures.
Failure to log off properly can allow unauthorized access from your workstation by another
user.
4.1.6 Protect your key fob from physical abuse. Do not immerse it in liquids or expose it to
extreme temperatures.
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Setting Up a New PIN
1. Open a web browser, either Netscape or Explorer.
2. Access the CDC SecurID System website by entering the following address for location:
http://securid.cdc.gov
3. Select Set new PIN.
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Assigned by CDC
4. Enter your Username:
You have received your username in a separate communication.
NOTE: Please be sure there are five or six “ticks” on the left side of the device. This will allow time for
synchronization between the device and the server for authentication.
5. Click Send.
If you receive this screen again, please wait for the next number on the SecurID Key Fob, then enter the
username and number.
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6. Enter the PASSCODE: Initially, this is the six digits displayed on the SecurID Key Fob.
7. Click Send.
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8. Enter a four digit number that will serve as your PIN.
9. Then enter your PIN again for verification.
10. Click Send.
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11. Your PIN has been accepted. Wait for the numbers on the SecurID Key Fob to change, then
enter your PASSCODE. (Now it is your four digit PIN + six digits displaying on SecurID Key
Fob – no spaces.)
12. Click Send.
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You have now set up and tested your PIN.
NOTE: Please be sure to read and observe Precautions for Protecting Your SecurID Key Fob and the
secured information you will be accessing.
NOTE: If you leave your current position, notify CDC PulseNet and return the SecurID Key Fob to
FedEx to the following address:
Attn: Michael Korth
Centers for Disease Control
1600 Clifton Road, NE
MS C03
Atlanta, GA 30333
Phone: (404) 639-3334
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5. FLOW CHART:
6. BIBLIOGRAPHY:
7. CONTACTS:
7.1 PulseNet Database Team
Centers for Disease Control and Prevention
1600 Clifton Road NE
MS C03
Atlanta, Georgia 30333
Phone: (404) 639-4558
Email: PFGE@cdc.gov
8. AMENDMENTS:
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1. PURPOSE: To describe the procedure for authenticating and connecting to the server.
2. SCOPE: This procedure applies to all PulseNet personnel who are analysis certified and receive a
SecurID Key Fob.
3. DEFINITIONS/TERMS:
3.1. SecureID Key Fob: Token that displays a six-digit passcode. When used in combination with a
four digit PIN number, allows access through the CDC firewall.
3.2. BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium.
4. RESPONSIBILITIES/PROCEDURE:
Step 1: Authenticate to the Firewall
Open a web browser, either Netscape or Internet Explorer
Access your bookmark name Authentication Form or enter the following address for location:
http://gateway-sdn.cdc.gov:900.
You will see this screen: Client Authentication Remote Service
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Enter your login. You will receive your username in a separate communication.
Click Submit.
You will see this screen: Client Authentication Remote Service
Enter your 10-diigit password:
four-digit PIN plus six-digit SecurID number
NOTE: Please be sure there are two or three “ticks” on the left side of the device. This will allow time for
sychronization between the device and the server for authentication.
Click Authentication.
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You will see this screen: Client Authentication Remote Service
Verify Standard Sign-on is selected.
Click Submit.
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You will see this screen: Client Authentication Remote Service
You should have at least one rule.
Minimize the screen
NOTE: IF YOU CLOSE THIS SCREEN YOU WILL TERMINATE YOUR CONNECTION TO THE
FIREWALL.
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Step 2: Using BioNumerics
BioNumerics
v3.0
Open BioNumerics
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Select the database you want to analyze (this could be Ecoli-client, Salmonella-client, Listeria-client,
Shigella-client, or Campylobacter-client).
Click Analyze
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The PulseNet logo will appear if you have installed the PulseNet customization files.
This screen tells you the total number of isolates currently in your database, then the individual number,
by pattern.
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To connect to the Online BioNumerics server:
Click Database
Select Connect to server…
REMINDER: YOU SHOULD STILL HAVE THE AUTHENTICATION
SCREEN MINIMIZED.
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Enter the IP address of the BioNumerics server. You have received this in a separate communication.
Verify the Port number is 7013
Click OK
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Enter the Login and Password to access the BioNumerics server. You have received this in a separate
communication.
Click OK
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You will see the IP address and the server login
You have now successfully connected to the BioNumerics Online server.
5. FLOW CHART:
6. BIBLIOGRAPHY:
7. CONTACTS:
7.1 CDC PulseNet Database Team
Centers for Disease Control and Prevention
1600 Clifton Road NE
Mailstop C03
Atlanta, Georgia 30333
Phone: (404) 639-4558
Email: PFGE@cdc.gov
8. AMENDMENTS:
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1. PURPOSE: To describe the procedure for training PulseNet personnel in PulseNet database
management and communication protocols.
2. SCOPE: This procedure applies to all PulseNet participants and hosts of PulseNet database training
courses.
3. DEFINITIONS/TERMS:
3.1 Host lab: Term used to describe a PulseNet laboratory that has been approved by CDC to host a
training course
3.2 Training personnel: Term used to describe PulseNet participant(s) who have been approved by
CDC to train other PulseNet participants
3.3 APHL: Association of Public Health Laboratories
3.4 SOP: Standard Operating Procedure
3.5 CDC: Centers for Disease Control and Prevention
3.6 TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics
3.7 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
4. RESPONSIBILITIES:
4.1 All PulseNet personnel are required to read the PulseNet QA/QC manual and all PulseNet SOPs.
4.2 At least one PulseNet participant from each participating PulseNet laboratory is required to
attend annual PulseNet update meetings and regional meetings when they occur.
4.3 All PulseNet personnel submitting TIFFs of PulseNet pathogens for submission to the national
databases must have at least a basic level of computer knowledge and be familiar with the
BioNumerics analysis software.
4.4 Host lab(s), APHL, and/or CDC will determine training needs of PulseNet participants.
4.5 All PulseNet personnel must be trained by a host lab or by other approved training personnel.
4.5.1 PulseNet database management and communication training covers analysis of PFGE
gels using BioNumerics software, how to access the PulseNet National Databases with
data-sharing tools, how to upload PFGE data to the national server, and how to
communicate with others in the PulseNet network and CDC.
4.6 Host labs and training personnel should adequately prepare participants in the PulseNet database
management and communication. When training is finished, the trainee(s) should be able to
submit PulseNet gels and bundle files for analysis certification (see SOP PNQ02).
4.7 Host labs and training personnel must be analysis-certified.
4.8 Host labs must put together training materials for the trainees.
4.9 PulseNet participants will then be evaluated through certification (PNQ02) and proficiency
testing (PNQ04).
5. PROCEDURE:
5.1 APHL and CDC continuously monitor PulseNet participant training needs.
5.2 Host labs and/or training personnel should work with trainees and/or APHL to determine a
feasible time and location for the course.
5.3 The following is a recommended procedure for hosting a PulseNet database management course:
5.3.1 The host lab should organize an agenda committee to create an agenda and a timeline of
organizational duties, obtain needed training materials, and arrange for IT support.
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5.3.2
It is recommended that each participant be provided, but not limited to, the following
items:
5.3.2.1
Participant and trainer contact information
5.3.2.2
Handouts of training presentations, preferably in an organized notebook format
5.3.2.3
CD containing the BioNumerics software (for use in class only)
5.3.2.4
CD containing PulseNet masterscripts (for use in class only)
5.3.2.5
Practice exercises and training TIFFs with all PulseNet organisms
5.3.2.6
Course evaluations
5.3.2.7
Course certificates to indicate successful completion of training
5.3.3 The committee may also be responsible for making lodging, transportation, and meal
arrangements for course participants.
5.3.4 It is recommended that training be carried out using E. coli or Salmonella unless another
PulseNet organism is specifically requested.
5.3.5 Refer to PND02 (SOP for TIFF Image Analysis) and PND03 (SOP for Accessing and
Using the PulseNet Listserv) in the preparation of materials. Host labs can also contact
the CDC PulseNet Database Team for training materials.
5.3.6 Refer to Appendix PND10-01 for recommended training topics, and to PND01 (SOP for
Computer Equipment and Supplies) for a list of necessary materials.
5.3.7 Assign training responsibilities to trainers.
5.3.8 It is recommended that there be one trainer per five participants and a maximum of two
participants per computer.
5.3.9 At the completion of training, participants should fill out evaluations and be awarded
certificates of completion.
5.3.10 Summarize evaluations and supply trainers and members of the agenda committee a
summary of the training evaluations.
6. FLOW CHART:
7. BIBLIOGRAPHY:
8. CONTACTS:
8.1 PulseNet Computer support CDC contact: Brenda Brown
(404) 639-3942
BLBrown1@cdc.gov
8.2 Training support at CDC: PulseNet Database Administration Team
(404) 639-4558
PFGE@cdc.gov
9. AMENDMENTS:
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Appendix PND10-01
Sample BioNumerics and PulseNet Masterscripts Training Course Agenda
SAMPLE AGENDA
Beginning BioNumerics Workshop for PulseNet Participants
General Information about the Workshop
Welcome and Introductions; Overview of Workshop
Overview of BioNumerics—Goals and Objectives
BioNumerics 3.0/3.5 and PulseNet Master Scripts
Beginning BioNumerics: Processing PFGE Gel Images
Exercise 1: Processing a PFGE gel image and linking entries to a database
Review of Morning Sessions: Questions and Answers
How to Setup Your SecurID Key Fob, Authenticating to CDC Firewall, and Using the
PulseNet BioNumerics Online Server
Uploading and Accessing Data via the National Server
Exercise 2: Uploading data to the National server
Questions and Answers
Exercise 3: Practice using Salmonella or E. coli clients to analyze and enter data
Review of Day 1: Questions and Answers
Basics behind Comparisons and Clustering
Creating Comparisons in BioNumerics
Exercise 4: Performing comparisons
Communication with PulseNet and WebBoard
Exercise 5: Pulling it all together: practice scenario
Questions and Answers, Return Evaluation Form,
Presentation of Certificates
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STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS
MASTERSCRIPTS FOR NEW PULSENET DATABASES
CODE: PND11
Effective Date:
9
24
2007
1. PURPOSE: To describe the procedure for beta testing BioNumerics MasterScripts for new PulseNet
databases.
2. SCOPE: This procedure applies to CDC staff and PulseNet laboratories outside CDC who participate
in beta testing exercises.
3. DEFINITIONS/TERMS:
3.1.
3.2.
3.3.
3.4.
3.5.
3.6.
3.7.
3.8.
3.9.
3.10.
3.11.
3.12.
3.13.
3.14.
3.15.
APHL: Association of Public Health Laboratories
SOP: Standard Operating Procedure
CDC: Centers for Disease Control and Prevention
TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics.
Bundle file: A file generated within BioNumerics for sharing and comparing PFGE patterns and
associated demographic and laboratory data from one database to another. The file extension is .bdl
and is located in the bundle folder of the corresponding database directory (X://Program
files/bionumerics/data/test_organism database/bundle)
BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths (Sint-MartensLatem, Belgium.
Scripts: Macros (computer commands) which allow a repeated task in BioNumerics to be
automated.
MasterScripts: Macros designed specifically for a PulseNet laboratory to submit and query PFGE
patterns and demographic data on a national database.
Database Team Members: personnel at CDC who have been designated to manage a particular
PulseNet database; these people manage all administrative aspects of the databases, including being
involved in script creation, testing, naming patterns, and correcting band markings.
On-line: Connected to the PulseNet server. Certified individuals may access the information within
the particular on-line database.
Test database: Term used to describe the new database that has been created in which to test the
MasterScripts for the new database.
Beta testing: Testing a pre-release version of the MasterScripts by making it available to selected
users.
Screen dump: By pressing the “print screen” key on a computer keyboard, a user captures an image
of whatever is currently displayed on the monitor. A user may then paste this image into another
program such as Microsoft PowerPoint.
PFGE inbox: An email account that is maintained and checked by all database team members at
CDC. The address is PFGE@cdc.gov.
Remote testing lab: A PulseNet-participating lab other than CDC that has been designated to
perform testing on new scripts.
4. RESPONSIBILITIES AND PROCEDURE:
4.1. Internal Testing to be completed at CDC by Database Team Member(s).
4.1.1. The Database Team Member(s) will use the Testing Results_Client and Testing Results
_Admin spreadsheets (excel files found in P:\BioNumerics\Scripts\Testing\Testing
Templates) as guidelines to complete the beta testing and record any errors or
comments as necessary. Any errors should be captured in a screen dump and explained
in a PowerPoint slide.
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STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS
MASTERSCRIPTS FOR NEW PULSENET DATABASES
CODE: PND11
Effective Date:
9
24
2007
4.1.2.
The Testing Results_Client and Testing Results_Admin spreadsheets and the
PowerPoint slides will be saved in P:\BioNumerics\Scripts\Testing\MasterScripts v##
testing and/or Organism Testing.
4.1.3. The Database Team Member(s) will notify the PulseNet Database Unit Chief and
PulseNet Computer Support once testing has been completed and the spreadsheets and
PowerPoint slides are ready for review.
4.1.4. PulseNet Computer Support will review the spreadsheets and address any script errors
or comments as necessary and fill in any corrective measures taken. Once this is
completed, the Database Team Member and Database Unit Chief are notified for
additional testing as necessary.
4.2. External testing is to be completed by a remote testing lab. This can be a laboratorian from a state
or local health department who has been analysis-certified by CDC for at least one organism (see
Certification SOP PNQ02).
4.3. At least one PulseNet participant from a remote testing lab and a Database Team Member should
beta test the MasterScripts before new MasterScripts are distributed to PulseNet participants and the
new database is considered “on-line” (if a new database was added to the MasterScripts) and
accessible to analysis-certified personnel.
4.4. Those performing testing at CDC will need to have login access to the test database. This is
accomplished by:
4.4.1. The Database Team Member(s) responsible for the test database initiates an e-mail to
PulseNet Computer Support or other responsible individual for request to access the on-line
test database for beta testing of new scripts.
4.4.2. PulseNet Computer Support will initiate access to the test database.
4.4.3. Login and password information will be given to the individual(s) who will be completing the
beta testing once approval for access has been granted.
4.5. The Database Unit Chief will forward all necessary documents and files needed for beta testing to
the remote testing lab. These documents and files include:
4.5.1. Cover letter (see Appendix PND11-1)
4.5.2. Instructions for setting up a new database in BioNumerics (taken from BioNumerics manual
section “Setup”)
4.5.3. Instructions for installing new scripts from the MasterScripts CD
4.5.4. CD containing the newest beta version of the MasterScripts
4.5.5. File(s) of testing PFGE gel(s) (.tif)
4.5.5.1. PFGE gel file name is BETA TEST GEL_organism.tif
4.5.6. PFGE report containing necessary demographic and laboratory data for testing PFGE gel(s)
4.5.7. Checklist for beta testing new MasterScripts (see Appendix PND11-2)
4.6. PulseNet participants in the remote testing lab should read and become familiar with instructions
for creating a new database and installing scripts prior to the start of beta testing.
4.7. The following procedure for beta testing scripts for a new PulseNet national database is as follows:
4.7.1. Follow instructions to create a new PulseNet database in BioNumerics. The name of the
database should be test_organism initials (i.e. test_EC).
4.7.2. Follow instructions to install MasterScripts from CD in BioNumerics
4.7.3. Analyze the test PFGE gel(s) in BioNumerics
4.7.4. Link lanes and enter demographic and laboratory data as described in the PFGE report(s)
4.7.5. Create a bundle file (.bdl) and e-mail it to the PFGE inbox with “Test Bundle” in the subject
line of the email.
4.7.5.1. This step ensures that the reference standard and entry property fields have been
installed correctly in the client database.
4.7.6. Once the bundle file has been sent, await confirmation from the PulseNet Database Team
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STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS
MASTERSCRIPTS FOR NEW PULSENET DATABASES
4.7.7.
4.7.8.
4.7.9.
4.7.10.
4.7.11.
4.7.12.
4.7.13.
4.7.14.
4.7.15.
4.7.16.
CODE: PND11
Effective Date:
9
24
2007
Create a comparison of all entries in the local database
Select the experiment type icon (e.g. PFGE-XbaI) on the bottom of the comparison window
to visualize the PFGE patterns. Note: All patterns should be visible and no remapping errors
should be shown.
Perform queries on your local test organism database. Make note of any errors or anything
that does not perform correctly.
If a new database has been added to the MasterScripts, look over the fields, pick lists, and
organization of the organism database. Make note of anything that appears incorrect and/or
list any ideas you have to improve the look or organization of the new database.
Perform any other queries or actions as directed by the PulseNet Database Team. This will
include any new items or tools added to the MasterScripts.
The checklist (Appendix PND11-2) should be completed and comments added and sent back
to the PFGE inbox.
The Database Team Member responsible for beta testing of the database should give
feedback and results to all participants involved with beta testing of MasterScripts within 2
weeks of the testing date.
If errors are found, the PulseNet Database Team will communicate those errors to PulseNet
Computer Support or Applied Maths for resolution.
Once all testing is successfully completed, the MasterScripts can be distributed and any new
databases should then become accessible on-line to all analysis-certified participants.
The certification process should be initiated for PulseNet laboratories interested in submitting
data.
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STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS
MASTERSCRIPTS FOR NEW PULSENET DATABASES
CODE: PND11
Effective Date:
9
24
2007
5. FLOW CHART:
Beta Testing MasterScripts
for New PulseNet
Databases
External Testing of
Client MasterScripts
Database Unit Chief asks various
labs to test MasterScripts
Internal Testing of
Client and Admin
MasterScripts
CDC Database Team
Member(s) test MasterScripts,
complete Testing Results
spreadsheets, and create
necessary PowerPoint slides to
show any errors
CDC Database Team
Member(s) notify Database
Unit Chief and PulseNet
Computer Support once
testing is completed
PulseNet Computer Support
reviews Testing Results
spreadsheets and addresses any
errors and notifies Database Unit
Chief and Database Team
Member(s) if additional testing is
necessary
Database Unit Chief sends a
cover letter, instructions, the CD
containing MasterScripts, testing
files (TIFF), and a checklist to
external testing labs
External labs follow the instructions for
installing and testing the new
MasterScripts. Once testing is
completed, the lab sends the checklist
with any errors and/or comments back
to CDC
PulseNet Computer Support
and the Database Team
Member(s) review the
checklists and address any
errors and/or comments
External testing is complete
once all errors have been
addressed
The new MasterScripts are
distributed to participating labs
Internal Testing is complete
once all errors have been
addressed
If applicable, new database
becomes accessible on-line
and certification sets are made
available
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STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS
MASTERSCRIPTS FOR NEW PULSENET DATABASES
CODE: PND11
Effective Date:
9
24
2007
6. BIBLIOGRAPHY:
7. CONTACTS:
7.1
CDC PulseNet Computer Support
Brenda Brown
(404) 639-3942
BLBrown1@cdc.gov
7.2
CDC PulseNet Database Team
(404) 639-4558
PFGE@cdc.gov
7.3
PulseNet Database Team Unit Chief
Kelley Hise
(404) 639-0704
KHise@cdc.gov
8. AMENDMENTS:
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STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS
MASTERSCRIPTS FOR NEW PULSENET DATABASES
CODE: PND11
Effective Date:
9
24
2007
Appendix PND11-1
Beta Testing MasterScripts Cover Letter Template
Beta testing organism MasterScripts vXX Beta
Scripts for PulseNet participating laboratories enable users to share and compare patterns within the
PulseNet community. Reference systems (H9812), pick lists, and other functions within BioNumerics are
standardized using scripts so that data is comparable among participating laboratories. These scripts are
developed by CDC and Applied Maths and are highly customized for each national database.
Before a set of scripts are implemented for use by the PulseNet community, a series of beta testing is done
at both CDC and remote PulseNet laboratories. These testing procedures test the functionality of the
database (i.e. ability to analyze and enter demographic/laboratory data, queries, etc.) as well as the overall
layout and look of the database. This testing is greatly needed and participation from PulseNet
laboratories is always appreciated.
This packet contains all the necessary information for remote PulseNet laboratories to beta test scripts for
new PulseNet national databases. This beta testing will be for the organism national database which is
considered online and in the production phase of testing. If at any time you have questions, please do not
hesitate to call or e-mail the PulseNet Database Team at 404-639-4558 or pfge@cdc.gov.
Below are the following documents and CD that are included in this packet:
1. CD containing 2 folders:
a. MasterScripts vXX Beta: has the files for testing organism scripts
b. Testing: has PFGE TIFFs of organism and TIFF demographic information, electronic copy of
checklist and procedures
2. Laboratory and demographic data for PFGE TIFFs
3. Checklist for testing the MasterScripts for the organism database
Thank you again for testing the MasterScripts vXX Beta. Your participation in PulseNet and in the testing
has been greatly appreciated.
PulseNet Database Team
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CODE: PND11
STANDARD OPERATING PROCEDURE FOR BETA TESTING BIONUMERICS
MASTERSCRIPTS FOR NEW PULSENET DATABASES
Effective Date:
9
24
2007
Appendix PND11-2
Checklist for testing MasterScripts vXX Beta for PulseNet
organism database
LabID____________
Date Testing Completed_______________
Insert an “X”
when
completed
Item to be Tested for organism
Create a local test organism database
Install MasterScripts vXX Beta for the test organism database
Analyze a organism PFGE gel (test of gel analysis)
Add demographic information for gel (test of pick lists, etc)
Create bundle file and e-mail to: pfge@cdc.gov with “Test Bundle” in subject line (test of
bundle file function)
Perform queries on your local test organism database
Create comparison and pull up patterns to view in comparison window
Look over the fields, pick lists and organization of the organism database (please comment
below)
Comments:
After completing, either fax [404-639-3333 (ATTN: PulseNet Database Team)] or email to
pfge@cdc.gov and put “Testing” in the subject line. Please don’t forget to fill in the LabID information at
the top.
Thank you.
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CODE: PND12
STANDARD OPERATING PROCEDURE FOR NAMING PULSENET OUTBREAKS
AND CLUSTERS
Effective Date:
4
01
2006
1.
PURPOSE: To describe the PulseNet standardized nomenclature for outbreaks and clusters.
2.
SCOPE: This procedure applies to all CDC PulseNet Database personnel entering outbreak and cluster
information into the “Outbreak” field in BioNumerics.
3.
DEFINITIONS/TERMS:
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
3.10
3.11
4.
TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics.
BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
CDC: Centers for Disease Control and Prevention
PFGE: Pulsed-field Gel Electrophoresis
Cluster: A group of isolates with the same serotype determined to possess indistinguishable PFGE DNA
patterns using one or more enzyme restrictions.
DNA: Deoxyribonucleic acid
LITS: Laboratory Information Tracking System
LabID: Unique identification assigned to each PulseNet participating laboratory, usually two-four letters
that correspond with the postal code that is assigned by CDC.
PulseNet Listserv: A closed, moderated, electronic web conference used for communication among
PulseNet participants. The PulseNet Listserv is open to all laboratory staff at PulseNet participating
laboratories. Epidemiologists at these laboratories, when approved by the PulseNet laboratory contact,
U.S. food regulatory staff, and PulseNet International representatives may also have access. The PulseNet
Listserv is not open to university or private industry personnel unless their inclusion is deemed to be in the
interest of public health.
Outbreak: A cluster of cases of infections with a common epidemiological exposure, e.g. to a specific food
product.
Cluster: A group of isolates with the same serotype with indistinguishable PFGE DNA patterns by one or
more restriction enzyme
RESPONSIBILITIES/PROCEDURE:
4.1 Basic code: A code consisting of the date of recognition of the cluster in the YYMM (2-digit year and 2digit month) format is created, followed by the LabID abbreviation where it was first recognized (via
PulseNet Listserv or other such posting).
<Date><LabID><LITS code for organism>-<number of cluster in month>
4.1.1
If a cluster is recognized at more than one lab simultaneously, the letter code should be “ml” for
multi location.
4.1.2
The reason for this order of characters in the basic code is so it will be possible to query and sort
the result of a query by year using only this single field.
4.2 The first characters identify the cluster/outbreak in a unique way. A dash (“-”) plus a digit should follow
the initial 9-11 characters (accounting for LabIDs with >2 characters) to indicate:
4.2.1
The number of the cluster/outbreak for the month. There can be different clusters/outbreaks with
different serotypes occurring simultaneously in the same state; hence, to avoid the risk of mixing
information on separate clusters/outbreaks together in the databases, the three-letter code for the
serotype used in the naming of the PFGE-profiles should also be added after the date information.
4.2.1.1 Example: 0805MAJEG-2 is the second Salmonella Enteritidis outbreak in Massachusetts, first
seen in May 2008. “0805” indicates the cluster was first noticed in May of 2008; “MA”
indicates that it was first seen in MA; “JEG” represents Salmonella Enteritidis; the “-2”
indicates that this is the second outbreak of this serotype for this month recognized in MA.
4.2.2
A “?” indicating that the status of the profile/case has not been firmly established as being
associated with the cluster
4.2.3
An “x” indicating isolates that are being used as controls in a study
4.2.4
Examples:
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STANDARD OPERATING PROCEDURE FOR NAMING PULSENET OUTBREAKS
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CODE: PND12
Effective Date:
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01
2006
4.2.4.1 0805mlGX6-1 (the first Listeria monocytogenes multi-state outbreak recognized
simultaneously in two or more states in May 2008)
4.2.4.2 0210MSJPX-3 (a cluster of cases associated with Salmonella Typhimurium discovered in
October 2002 following two other clusters/outbreaks in Mississippi that month
4.2.4.3 0207FLJ16-2? The second Shigella sonnei Florida outbreak turns out to be a big one and
there are a lot of isolates that may be related to the outbreak. It takes time to sort out whether
they are part of the outbreak (the database manager has received a lot of TIFFs or they have
done a search on the profile but haven’t analyzed the results, yet). It could also be a single
unconfirmed isolate from a patient in a different state in a presumably single state outbreak.
4.2.4.4 0309WADBR-1? This is a big one, recognized as a cluster of Campylobacter jejuni in
Washington in September of 2003. Before they get interrupted, the database manager names
all the possible matches they have found so that they may find them again easily.
4.2.4.5 0307GAJJP-1x This is the first outbreak of Salmonella Newport that began in July of 2003 in
Georgia; however, the “x” on the end denotes that these were considered to be controls in the
outbreak, and do not have the outbreak pattern. This simplifies being able to pull these up at a
later date.
5.
FLOW CHART:
6.
BIBLIOGRAPHY:
7.
CONTACTS:
8.
AMENDMENTS:
8.1 2009-01-01: Removed the “c” that used to be placed at the end of the basic outbreak code indicating the
cluster was not yet an outbreak. Also removed the “ml” that used to be placed at the end of the basic outbreak
code indicating the cluster was a multi-state cluster.
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PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157
(STEC O157) IN BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND14
Effective Date:
05
11
07
1. PURPOSE: to describe the standardized protocol for analysis of MLVA data of
Shiga toxin-producing Escherichia coli O157 (STEC O157) in BioNumerics.
2. SCOPE: to provide PulseNet participants with a single protocol for analyzing MLVA
data of STEC O157, thus ensuring inter-laboratory comparability of the generated
results.
3. DEFINITIONS:
3.1 MLVA: Multiple-locus variable-number tandem repeat analysis
3.2 VNTR: Variable-number tandem repeat
3.3 CDC: Centers for Disease Control and Prevention
3.4 SOP: Standard Operating Procedure
4. RESPONSIBILITIES/PROCEDURE:
4.1 Software needed for data analysis
4.1.1. BioNumerics version 3.0 or higher
4.1.2. Customized scripts for data import (VNTRImport_v4.bns), copy number
calculation (= allele assignment) (VNTRCalc_v4.bns) and troubleshooting
(VNTRReport.bns). An additional script (VNTRDetails_v2.bns) facilitates
special functions such as calculation of diversity indices.
4.1.3 Look-up table (BeckmanEcoli.txt) for allele size ranges
4.2 General overview
4.2.1 The import process consists of three major steps:
4.2.1.1 Exporting the appropriate data file from the Beckman system (refer
to SOP PNL19)
4.2.1.2 Importing this file into BioNumerics (using the script
VNTRImport_v4.bns)
4.2.1.3 Determining the copy numbers (assigning alleles) for each VNTR
(using the script VNTRCalc_v4.bns and the look-up table
BeckmanEcoli.txt)
4.2.1.3.1 VNTRCalc_v4.bns script facilitates the allele assignment in
two different ways:
4.2.1.3.1.1 “BeckmanEcoli” – assigns the copy number based on
the look-up table. This method should be used in all
routine analysis.
4.2.1.3.1.2 “Predicted” – calculates the copy number based on the
mathematical formula: (Observed fragment size –
offset)/repeat size. This method should only be used
when a new allele (not specified in the look-up table) is
identified
4.3
Required import format
4.3.1 The data should be exported from the Beckman software as a CSV
(comma-delimited) text file containing the fragment length information.
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PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157
(STEC O157) IN BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND14
Effective Date:
05
11
07
Multiple columns will be present in the file but only four columns are of
interest: “RN,” “dye,” “est size (nt)” and “pk height (rfu)”. All other
columns will be ignored by the script during import. The field “RN”
should contain information designating the BioNumerics key number
(isolate identifier) loaded in each lane of the sequencer, as well as which
VNTR mastermix or reaction was loaded. The import script will ignore
any text that appears beyond the dot “.” in the RN field.
4.4 Setting up a new database
4.4.1 Click “New” to set up a new database
4.4.2 Type in the name (for example “O157MLVA”) of your new database and
click ”Next”
4.4.3 Select the default directory to set up a new database and click “Next”
4.4.4 Choose “Yes” to enable creation of log files and click “Finish”
4.4.5 Choose the default “Use the local database” and click “OK”
4.4.6 Go to: Program files → BioNumerics → Data → O157MLVA and create
two folders named “Scripts” and “VNTRtables”.
4.4.7 Save the four MLVA specific scripts (VNTRImport_v4.bns,
VNTRCalc_v4.bns, VNTRReport.bns, VNTRDetails_v2.bns) in the
“Scripts” folder and the look-up table (BeckmanEcoli.txt) in the
“VNTRtables” folder
4.5 Importing a peak file for the first time
4.5.1 Review the peak file: make sure that the observed size for the D1 labelled
molecular size standard peaks is within ± 1 bp from the expected size.
Remove any data (failed reactions, controls, internal ladder) that you don’t
want to import in the BioNumerics from the CSV file. Re-name and resave the CSV file either on your hard drive or on the flash drive.
4.5.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “Beckman peak file.” The
script pops up a file dialog box, prompting for the name of the file to
import. Select the appropriate file and click “OK.” A second dialog box
pops up, prompting for a variety of other information:
4.5.2.1 Fingerprint file name. This is the name of the fingerprint file that will
be used in BioNumerics. Leave this unchanged, unless a file has
already been imported with the same name. In this case, change the
file name into a new, unique and informative name.
4.5.2.2 Dyes to import. The first dye (D1) contains only reference markers and
does not need to be imported. Use this list to select what dyes will be
imported; they are D2, D3 and D4. Each dye will be stored in a
different file, resulting from appending the dye name to the fingerprint
file name.
4.5.2.3 Assign reference positions. Leave this option checked.
4.5.2.4 Select imported isolates. Leave this option checked.
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PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157
(STEC O157) IN BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND14
Effective Date:
05
11
07
4.5.2.5 Pool tags. For this protocol, there are two pool tags representing the
two PCR reactions for each isolate (all loaded on different wells) and
tagged with names “R1” and “R2”. The two tags should appear in the
list. If they do not, they can be added by selecting “Add” to update.
4.5.2.6 If everything is filled appropriately, click “OK” to start the import.
4.5.2.7 NOTE: These settings are automatically saved and reloaded the next
time this script is run.
4.5.3 When the script is finished, it has:
4.5.3.1 Created a fingerprint type “VNTRFpr” with appropriate settings
4.5.3.2 Created fingerprint types with appropriate settings for each dye and
each pool used. The names of these fingerprint types are
“VNTRFpr”+“pool name”+“dye” (for example: VNTRFprR1_D2).
4.5.3.3 Created fingerprint files for each dye in the fingerprint type
“VNTRFpr,” and imported the band information in these lanes. The
individual lanes are assigned to their appropriate pool fingerprint type,
according to the known pool tags found in the “RN” field.
4.5.3.4 Created new database entries for all isolates found
4.5.3.5 Linked the fingerprints to their corresponding entries
4.5.3.6 Selected all isolates for which fingerprint data was imported
4.6 Determining the copy numbers for the first time (allele assignment)
4.6.1 Run the script VNTRCalc_v4 from the scripts menu. The first time this
script is run, it will automatically create a new character set called
“VNTR_vals”. This character set will hold the copy numbers for each
VNTR. The script will also create a second character set called
“VNTR_frags” which holds the fragment size with a highest fluorescence
for each VNTR. Click “OK” on the two pop-up windows notifying that
these two character types will be created.
4.6.2 Before the script can determine copy numbers from the fragment length
information, it should have sufficient information about what VNTR’s can
be found in which pool and dye, what are the repeat lengths, etc. To this
end, the script pops up a dialog box called “VNTR assign”. This dialog
box contains a list of defined VNTR’s that is initially empty. To enter the
parameters of the first VNTR, click on “Add”. This brings up a second
dialog box, prompting for all properties of this first VNTR. Refer to the
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(STEC O157) IN BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND14
Effective Date:
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laboratory protocol PNL19, appendix PNL19-4 for all necessary
information to load the specifications for each VNTR.
4.6.3 The VNTR information
4.6.3.1 Name. This name will be used for further reference and for the
character name in the character type VNTR_vals. Each VNTR should
have a unique name (for example VNTR_3, VNTR_34, etc).
4.6.3.2 Offset. Each fragment consists of a repeat portion and a constant
portion, due to the fact that the primers do not occur exactly at the start
and the end of the repeat region. This parameter specifies the size (in
base pairs) of the constant portion.
4.6.3.3 Repeat length. This parameter specifies the size of a unit repeat block
(in base pairs).
4.6.3.4 Copy range. These parameters specify the minimum and maximum
number of copies that the script will consider during the copy number
determination.
4.6.3.5 Tolerance. This specifies the maximum difference between the
expected fragment length (calculated by the software) and the actual
length estimated by the sequencer.
4.6.3.6 Take from fingerprint type. This drop-down list should be used to
indicate on what fingerprint type this VNTR was run. The fingerprint
type is determined by the dye and the pool tag.
4.6.4 If all parameters are filled in appropriately, click OK to add the VNTR and
return to the previous dialog box (“VNTR assign”). Repeat the same
actions to enter the information for all VNTRs used for this protocol.
When all VNTRs are entered, make sure the box for “Code absent as
negative” is checked. Select “BeckmanEcoli” from the “Fragment ranges”
dropdown menu and press the “Save&Assign” button to let the script
assign copy numbers for the currently selected entries.
4.6.4.1 NOTE 1: pressing “Save&Quit” would store the information without
calculating copy numbers for the current selection.
4.6.4.2 NOTE 2: all information regarding the VNTR’s is stored with the
database. The next time the script is run, the VNTR definitions will be
loaded automatically. New incoming VNTR data can be processed
simply by selecting the corresponding data entries, launching the script
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PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157
(STEC O157) IN BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND14
Effective Date:
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VNTRCalc_v4 from the “Scripts” drop-down menu, and pressing the
button “Save&Assign.”
4.6.4.3 For every fragment with the highest fluorescence level in the
fingerprint type (= the combination of the reaction and the dye), the
script will assign an allele type (=a copy number) based on the
fragment size ranges specified in the “BeckmanEcoli” look-up table
(attachment PND-1). Note that, within the same fingerprint type, more
than one VNTR can be loaded if there is no overlap within the
fragment size ranges.
4.6.5 When the script has completed, all copy numbers for all VNTR’s for the
currently selected entries are determined.
4.6.5.1 Note: Two types of problems may arise during the process (if one or
more such errors were encountered during the calculations, an error
report is displayed listing all the problems):
4.6.5.1.1 None of the peaks present in a fingerprint are compatible with
the fragment size ranges in the look-up table. In this case, the
corresponding character value will be scored “-2.0”. In this
situation, run the “VNTRReport” script from the “Scripts”
drop-down menu to verify whether the problem occurred
because the VNTR failed to amplify (mutations, insertions or
deletions in the primer annealing region) or because the VNTR
had a fragment size outside the acceptable range. In the latter
case, rerun the reaction to confirm the accuracy and
reproducibility of the sizing. The isolate should be submitted to
CDC for confirmation if the size upon retesting is still outside
the fragment range specified in the look-up table.
4.6.5.1.2 More than one peak in a fingerprint is compatible with an
acceptable fragment size range. In this case, the script will use
the solution that corresponds to the peak with the largest “peak
height” value.
4.7 Importing a peak file on a routine basis
4.7.1 Review the peak file: make sure that the observed size for the D1 labelled
molecular size standard peaks is within ± 1 bp from the expected size.
Remove any data (failed reactions, controls, internal ladder) that you don’t
want to import in the BioNumerics from the CSV file. Re-name and resave the CSV file either on your hard drive or on the flash drive.
4.7.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “Beckman peak file”. The
script pops up a file dialog box, prompting for the name of the file to
import. Select the appropriate file and click “OK”. “VNTR/Import peak
data” dialog box will appear. De-select “D1” from the “Dyes to import”
and click “OK”.
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PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157
(STEC O157) IN BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND14
Effective Date:
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4.7.3
Run the script VNTRCalc_v4 from the scripts menu. “VNTR assign”
dialog box will appear. Select “BeckmanEcoli” from the “Fragment
ranges” dropdown menu and click “Save&Assign” to assign allele
numbers.
4.8 Verifying the allele assignment
4.8.1 For each isolate in the database that has some VNTR data associated, you
can click on the “VNTR frags” and “VNTR vals” entries in the list to open
the “Entry edit” windows.
4.8.2
If the VNTRCalc_v4 script did not detect a fragment for a VNTR, refer to
the step 4.6.5.1.1 of this protocol for details on how to proceed. If the
fragment size is slightly outside the range specified in the “BeckmanEcoli”
look-up table it is possible to manually assign a temporary allele size and
type for that VNTR until an official confirmation has been performed by
CDC (Note: only the CDC database managers are allowed to modify the
BeckmanEcoli.txt file. Once a modification has been made, the modified file
will be posted on CDCTeamSupport along with a notification that the file
has been modified). To manually assign an allele size and type click on the
fragment size in the “VNTRfrags” entry edit window or the allele type in
the “VNTR vals” entry edit window. This will open the “Change character
value” window in BioNumerics lower than 5.0. In BioNumerics 5.0 the
allele size or type can be directly highlighted and changed in the entry edit
window (Screen shots below)
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MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157
(STEC O157) IN BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND14
Effective Date:
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4.9 Visualizing the data
4.9.1 In order to display copy numbers next to a dendrogram in a comparison,
first create a “composite data set” that holds the VNTR data.
4.9.1.1 From the “Experiments” drop-down menu, select the option “Create
new composite data set…”, enter a name (e.g. VNTR_cmp), and click
the “OK” button. The “Composite data set ‘VNTR_cmp’” window
will appear.
4.9.1.2 Highlight the experiment VNTR_vals and from the “Experiment” dropdown menu, select the option “Use in composite data set”. Close the
window.
4.9.2 Next time a comparison window is opened (see below “Analysis of the
VNTR data”), there will be a new experiment VNTR_cmp listed in the
bottom of the window (BioNumerics versions lower than 5.0) or in the top
left corner of the window (BioNumerics version 5.0). This experiment will
facilitate the display of a spreadsheet-like view of the copy numbers (note
that it may be necessary to scroll the experiment list to the right with the
arrow button to bring VNTR_cmp in display in BioNumerics versions
lower than 5.0). This can be shown next to a dendrogram analysis of the
data set.
4.10 Analysis of the VNTR data
4.10.1. The VNTR data contained in the character set VNTR_vals can be analysed
in BioNumerics with all the tools that are available to character data. That
includes cluster analysis with a variety of methods and similarity
coefficients. For VNTR data, the coefficients that make most sense are:
4.10.1.1 Categorical: preferred if differences in copy numbers should be
treated in a qualitative way. This is the only option for creating
dendrograms using MLVA data.
4.10.1.2 Manhattan: preferred if differences in copy numbers should be treated
in a quantitative way (larger difference means more distantly related
organisms). This coefficient can be used to construct minimum
spanning trees.
4.10.2 In order to create a dendrogram:
4.10.2.1 Select the isolates to be included in the dendrogram
4.10.2.2 From the “Comparison” drop-down menu, select the option “Create
new comparison” and a “Comparison” window will appear
4.10.2.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics
versions lower than 5.0 or from the top left “Experiments” window in
BioNumerics 5.0.
4.10.2.4 From the “Clustering” drop-down menu, select the option
“Calculate…Cluster analysis (similarity matrix)” and a “Composite
data set comparison” dialog box will appear
4.10.2.5 Select “Categorical” for “Multi-state coefficient” and “UPGMA” for
“Dendrogram type”
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CODE: PND14
Effective Date:
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4.10.2.6 Click on “OK” button to finish the calculations and the “Comparison”
window with the dendrogram will reappear
4.10.2.7 From the “Layout” drop-down menu, select the option “Show image”
4.10.2.8 From the “Composite” drop-down menu, select the option ”Show
quantification (values)” and the copy numbers will appear next to the
dendrogram
4.10.3 In order to create a minimum spanning tree:
4.10.3.1 Select the isolates to be included in the spanning tree
4.10.3.2 From the “Comparison” drop-down menu, select the option “Create
new comparison” and a “Comparison” window will appear
4.10.3.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics
versions lower than 5.0 or from the top left “Experiments” window in
BioNumerics 5.0.
4.10.3.4 From the “Clustering” drop-down menu, select the option
“Calculate…Minimum spanning tree (population modeling)” and a
“Minimum spanning tree” dialog box will appear
4.10.3.5 Make sure the default “Manhattan” is checked for “Coefficient” and
click “OK” and the “Minimum spanning tree” window with the tree
will appear.
4.10.3.6 You can find out the content for each node by clicking on them
individually.
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(STEC O157) IN BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND14
Effective Date:
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5. FLOW CHART:
6. BIBLIOGRAPHY:
7. CONTACTS:
7.1. Eija Hyytia-Trees, D.V.M., Ph.D.
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-3672
EHyytia-Trees@cdc.gov
7.2. Patti Lafon
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-2828
PLafon@cdc.gov
7.3. Kara Cooper, Ph.D.
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-4358
KCooper@cdc.gov
8. AMENDMENTS:
2009-01-27: the allele (= copy number) assignment will be based on a look-up table
listing the actual observed fragment size ranges instead of using the mathematical
formula that assigns alleles based on predicted fragment sizes. The look-up table
approach in allele assignment will facilitate the comparison of fragment size data from
the Beckman Coulter CEQ 8000 and the Applied Biosystems Genetic Analyzer 3130xl in
the same database.
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MLVA DATA OF SHIGA TOXIN-PRODUCING ESCHERICHIA COLI O157
(STEC O157) IN BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND14
Effective Date:
05
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Appendix PND14-1
Fragment ranges for the STEC O157 VNTR alleles (BeckmanEoli look-up table)
Count
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
VNTR_3
333-334
339-340
345-346
350-353
356-358
362-364
368-370
374-377
380-383
386-389
392-395
398-401
404-407
410-413
417-419
423-425
429-431
435-437
442-443
448-449
454-456
460-464
466-468
VERSION:
VNTR_34
170-172
188-190
206-209
224-227
241-245
260-262
278-280
295-297
VNTR_9
471-472
482-484
488-489
494-497
499-502
507-509
512-515
518-521
525-527
530-533
536-539
543-546
549-551
555-558
561-563
566-570
572-576
579-581
584-586
590-592
597-598
603-604
608-611
614-615
REPLACED BY:
VNTR_25
116-119
122-124
128-130
135-136
141-142
146-148
153-154
158-160
165-166
170-171
188-190
VNTR_17
VNTR_19
130-133
137-140
143-146
150-152
156-159
162-165
169-172
175-178
182-184
188-190
195-197
199-201
282-284
291-292
296-299
302-304
307-311
314-316
320-321
326-328
332-333
338-339
344-345
VNTR_36
123-125
130-132
136-139
143-146
150-153
158-161
165-167
172-174
179-181
187-188
193-195
201-202
207-209
VNTR_37
164-166
171-172
177-178
183-185
188-191
195-197
200-203
207-209
213-215
219-220
225-226
231-232
237-238
241-244
227-228
273-274
418-419
AUTHORIZED BY:
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PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SALMONELLA ENTERICA SEROTYPE TYPHIMURIUM IN
BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND15
Effective Date:
07
05
11
1. PURPOSE: to describe the standardized protocol for analysis of MLVA data of
Salmonella enterica serotype Typhimurium in BioNumerics.
2. SCOPE: to provide PulseNet participants with a single protocol for analyzing MLVA
data of S. enterica serotype Typhimurium, thus ensuring inter-laboratory
comparability of the generated results.
3. DEFINITIONS:
3.1 MLVA: Multiple-locus variable-number tandem repeat analysis
3.2 VNTR: Variable-number tandem repeat
3.3 CDC: Centers for Disease Control and Prevention
3.4 SOP: Standard Operating Procedure
4. RESPONSIBILITIES/PROCEDURE:
4.1 Software needed for data analysis
4.1.1. BioNumerics version 3.0 or higher
4.1.2. Customized scripts for data import (VNTRImport_v4.bns), copy number
calculation (= allele assignment) (VNTRCalc_v4.bns), troubleshooting
(VNTRReport.bns), and support functions (VNTRDetails_v2.bns)
4.1.3 Look-up table (BeckmanST.txt) for allele size ranges
4.2 General overview
4.2.1 The import process consists of three major steps:
4.2.1.1 Exporting the appropriate data file from the Beckman system (refer
to SOP PNL21)
4.2.1.2 Importing this file into BioNumerics (using the script
VNTRImport_v4.bns)
4.2.1.3 Determining the copy numbers (assigning alleles) for each VNTR
(using the script VNTRCalc_v4.bns and the look-up table
BeckmanST.txt)
4.2.1.3.1 VNTRCalc_v4.bns script facilitates the allele assignment in
two different ways:
4.2.1.3.1.1 “BeckmanST” – assigns the copy number based on the
look-up table (PND15-1). This method should be used
in all routine analysis.
4.2.1.3.1.2 “Predicted” – calculates the copy number based on the
mathematical formula: (Observed fragment size –
offset)/repeat size. This method should only be used
when a new allele (not specified in the look-up table) is
identified
4.3 Required import format
4.3.1 The data should be exported from the Beckman software as a CSV
(comma-delimited) text file containing the fragment length information.
Multiple columns will be present in the file but only four columns are of
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BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND15
Effective Date:
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interest: “RN”, “dye”, “est size (nt)” and “pk height (rfu)”. All other
columns will be ignored by the script during import. The field “RN”
should contain information designating the BioNumerics key number
(isolate identifier) loaded in each lane of the sequencer, as well as which
VNTR mastermix or reaction was loaded. The import script will ignore
any text that appears beyond the dot “.” in the RN field.
4.3.1.1 NOTE: no spaces or underscores are allowed between the strain ID
and the reaction ID or between the reaction ID and the dot “.” in the
RN field.
4.4 Setting up a new database
4.4.1 Click “New” to set up a new database
4.4.2 Type in the name (for example “STMLVA”) of your new database and
click ”Next”
4.4.3 Select the default directory to set up a new database and click “Next”
4.4.4 Choose “Yes” to enable creation of log files and click “Finish”
4.4.5 Choose the default “Use the local database” and click “OK”
4.4.6 Go to: Program files → BioNumerics → Data → STMLVA and create
two folders named “Scripts” and “VNTRtables”.
4.4.7 Save the four MLVA specific scripts (VNTRImport_v4.bns,
VNTRCalc_v4.bns, VNTRReport.bns, VNTRDetails_v2.bns) in the
“Scripts” folder and the look-up table (BeckmanST.txt) in the
“VNTRtables” folder
4.5 Importing a peak file for the first time
4.5.1 Review the peak file: make sure that the observed size for the D1 labelled
molecular size standard peaks is within ± 1 bp from the expected size.
Remove any data (failed reactions, controls, internal ladder) that you don’t
want to import in the BioNumerics from the CSV file. Re-name and resave the CSV file either on your hard drive or on the flash drive.
4.5.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “Beckman peak file”. The
script pops up a file dialog box, prompting for the name of the file to
import. Select the appropriate file and click “OK”. A second dialog box
pops up, prompting for a variety of other information:
4.5.2.1 Fingerprint file name. This is the name of the fingerprint file that will
be used in BioNumerics. Leave this unchanged, unless a file has
already been imported with the same name. In this case, change the
file name into a new, unique and informative name.
4.5.2.2 Dyes to import. The first dye (D1) contains only reference markers and
does not need to be imported. Use this list to select what dyes will be
imported; they are D2, D3 and D4. Each dye will be stored in a
different file, resulting from appending the dye name to the fingerprint
file name.
4.5.2.3 Assign reference positions. Leave this option checked.
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BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND15
Effective Date:
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4.5.2.4 Select imported isolates. Leave this option checked.
4.5.2.5 Pool tags. For this protocol, there are two pool tags representing the
two PCR reactions for each isolate (all loaded on different wells) and
tagged with names “R1” and “R2”. The two tags should appear in the
list. If they do not, they can be added by selecting “Add” to update.
4.5.2.6 If everything is filled appropriately, click “OK” to start the import.
4.5.2.7 NOTE: These settings are automatically saved and reloaded the next
time this script is run.
4.5.3 When the script is finished, it has:
4.5.3.1 Created a fingerprint type “VNTRFpr” with appropriate settings
4.5.3.2 Created fingerprint types with appropriate settings for each dye and
each pool used. The names of these fingerprint types are
“VNTRFpr”+“pool name”+“dye” (for example: VNTRFprR1_D2).
4.5.3.3 Created fingerprint files for each dye in the fingerprint type
“VNTRFpr,” and imported the band information in these lanes. The
individual lanes are assigned to their appropriate pool fingerprint type,
according to the known pool tags found in the “RN” field.
4.5.3.4 Created new database entries for all isolates found
4.5.3.5 Linked the fingerprints to their corresponding entries
4.5.3.6 Selected all isolates for which fingerprint data was imported
4.6 Determining the copy numbers for the first time (allele assignment)
4.6.1 Run the script VNTRCalc_v4 from the scripts menu. The first time this
script is run, it will automatically create a new character set called
“VNTR_vals”. This character set will hold the copy numbers for each
VNTR. The script will also create a second character set called
“VNTR_frags” which holds the fragment size with a highest fluorescence
for each VNTR. Click “OK” on the two pop-up windows notifying that
these two character types will be created.
4.6.2 Before the script can determine copy numbers from the fragment length
information, it should have sufficient information about what VNTR’s can
be found in which pool and dye, what are the repeat lengths, etc. To this
end, the script pops up a dialog box called “VNTR assign”. This dialog
box contains a list of defined VNTR’s that is initially empty. To enter the
parameters of the first VNTR, click on “Add”. This brings up a second
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BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND15
Effective Date:
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dialog box, “Edit VNTR”, prompting for all properties of this first VNTR.
Refer to the laboratory protocol PNL21, appendix PNL21-4 for all
necessary information to load the specifications for each VNTR.
4.6.3 The VNTR information
4.6.3.1 Name. This name will be used for further reference and for the
character name in the character type VNTR_vals. Each VNTR should
have a unique name (for example ST3, ST5, etc).
4.6.3.2 Offset. Each fragment consists of a repeat portion and a constant
portion, due to the fact that the primers do not occur exactly at the start
and the end of the repeat region. This parameter specifies the size (in
base pairs) of the constant portion.
4.6.3.3 Repeat length. This parameter specifies the size of a unit repeat block
(in base pairs).
4.6.3.4 Copy range. These parameters specify the minimum and maximum
number of copies that the script will consider during the copy number
determination.
4.6.3.5 Tolerance. This specifies the maximum difference between the
expected fragment length (calculated by the software) and the actual
length estimated by the sequencer.
4.6.3.6 Take from fingerprint type. This drop-down list should be used to
indicate on what fingerprint type this VNTR was run. The fingerprint
type is determined by the dye and the pool tag.
4.6.4 If all parameters are filled in appropriately, click OK to add the VNTR and
return to the previous dialog box (“VNTR assign”). Repeat the same
actions to enter the information for all VNTRs used for this protocol.
When all VNTRs are entered, make sure the box for “Code absent as
negative” is checked. Select “BeckmanST” from the “Fragment ranges”
dropdown menu and press the “Save&Assign” button to let the script
assign copy numbers for the currently selected entries.
4.6.4.1 NOTE 1: pressing “Save&Quit” would store the information without
assigning copy numbers for the current selection.
4.6.4.2 NOTE 2: all information regarding the VNTR’s is stored with the
database. The next time the script is run, the VNTR definitions will be
loaded automatically. New incoming VNTR data can be processed
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BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND15
Effective Date:
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simply by selecting the corresponding data entries, launching the script
VNTRCalc_v4 from the “Scripts” drop-down menu, and pressing the
button “Save&Assign”.
4.6.4.3 For every fragment with the highest fluorescence level in the
fingerprint type (= the combination of the reaction and the dye), the
script will assign an allele type (=a copy number) based on the
fragment size ranges specified in the “BeckmanST” look-up table
(attachment PND15-1). Note that, within the same fingerprint type,
more than one VNTR can be loaded if there is no overlap within the
fragment size ranges.
4.6.5 When the script has completed, all copy numbers for all VNTR’s for the
currently selected entries are determined.
4.6.5.1 Note: Two types of problems may arise during the process (if one or
more such errors were encountered during the assignment, an error
report is displayed listing all the problems):
4.6.5.1.1 None of the peaks present in a fingerprint are compatible with
the fragment size ranges in the look-up table. In this case, the
corresponding character value will be scored “-2.0”. In this
situation, run the “VNTRReport” script from the “Scripts”
drop-down menu to verify whether the problem occurred
because the VNTR failed to amplify (mutations, insertions or
deletions in the primer annealing region) or because the VNTR
had a fragment size outside the acceptable range. In the latter
case, rerun the reaction to confirm the accuracy and
reproducibility of the sizing. The isolate should be submitted to
CDC for confirmation if the size upon retesting is still outside
the fragment range specified in the look-up table.
4.6.5.1.2 More than one peak in a fingerprint is compatible with an
acceptable fragment size range. In this case, the script will use
the solution that corresponds to the peak with the largest “peak
height” value.
4.7 Importing a peak file on a routine basis
4.7.1 Review the peak file: make sure that the observed size for the D1 labelled
molecular size standard peaks is within ± 1 bp from the expected size.
Remove any data (failed reactions, controls, internal ladder) that you don’t
want to import in the BioNumerics from the CSV file. Re-name and resave the CSV file either on your hard drive or on the flash drive.
4.7.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “Beckman peak file”. The
script pops up a file dialog box, prompting for the name of the file to
import. Select the appropriate file and click “OK”. “VNTR/Import peak
data” dialog box will appear. De-select “D1” from the “Dyes to import”
and click “OK”.
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4.7.3
Run the script VNTRCalc_v4 from the scripts menu. “VNTR assign”
dialog box will appear. Select “BeckmanST” from the “Fragment ranges”
dropdown menu and click “Save&Assign” to assign allele numbers.
4.8 Verifying the allele assignment
4.8.1 For each isolate in the database that has some VNTR data associated, you
can click on the “VNTR frags” and “VNTR vals” entries in the list to open
the “Entry edit” windows.
4.8.2
If the VNTRCalc_v4 script did not detect a fragment for a VNTR, refer to
the step 4.6.5.1.1 of this protocol for details on how to proceed. If the
fragment size is slightly outside the range specified in the “BeckmanST”
look-up table it is possible to manually assign a temporary allele size and
type for that VNTR until an official confirmation has been performed by
CDC (Note: only the CDC database managers are allowed to modify the
BeckmanST.txt file. Once a modification has been made, the modified file
will be posted on CDCTeamSupport along with a notification that the file
has been modified). To manually assign an allele size and type click on the
fragment size in the “VNTRfrags” entry edit window or the allele type in
the “VNTR vals” entry edit window. This will open the “Change character
value” window in BioNumerics lower than 5.0. In BioNumerics 5.0, the
allele size or type can be directly highlighted and changed in the entry edit
window (Screen shots below)
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4.9 Visualizing the data
4.9.1 In order to display copy numbers next to a dendrogram in a comparison,
first create a “composite data set” that holds the VNTR data.
4.9.1.1 From the “Experiments” drop-down menu, select the option “Create
new composite data set…”, enter a name (e.g. VNTR_cmp), and click
the “OK” button. The “Composite data set ‘VNTR_cmp’” window
will appear.
4.9.1.2 Highlight the experiment VNTR_vals and from the “Experiment” dropdown menu, select the option “Use in composite data set”. Close the
window.
4.9.2 Next time a comparison window is opened (see below “Analysis of the
VNTR data”), there will be a new experiment VNTR_cmp listed in the
bottom of the window (BioNumerics versions lower than 5.0) or in the top
left corner of the window (BioNumerics version 5.0). This experiment will
facilitate the display of a spreadsheet-like view of the copy numbers that
can be shown next to a dendrogram analysis of the data set.
4.9.2.1 NOTE: it may be necessary to scroll the experiment list to the right
with the arrow button to bring VNTR_cmp in display in BioNumerics
versions lower than 5.0.
4.10 Analysis of the VNTR data
4.10.1. The VNTR data contained in the character set VNTR_vals can be analysed
in BioNumerics with all the tools that are available to character data. That
includes cluster analysis with a variety of methods and similarity
coefficients. For VNTR data, the coefficients that make most sense are:
4.10.1.1 Categorical: preferred if differences in copy numbers should be
treated in a qualitative way. This is the only option for creating
dendrograms using MLVA data.
4.10.1.2 Manhattan: preferred if differences in copy numbers should be treated
in a quantitative way (larger difference means more distantly related
organisms). This coefficient can be used to construct minimum
spanning trees.
4.10.2 In order to create a dendrogram:
4.10.2.1 Select the isolates to be included in the dendrogram
4.10.2.2 From the “Comparison” drop-down menu, select the option “Create
new comparison” and a “Comparison” window will appear
4.10.2.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics
versions lower than 5.0 or from the top left “Experiments” window in
BioNumerics 5.0.
4.10.2.4 From the “Clustering” drop-down menu, select the option
“Calculate…Cluster analysis (similarity matrix)” and a “Composite
data set comparison” dialog box will appear
4.10.2.5 Select “Categorical” for “Multi-state coefficient” and “UPGMA” for
“Dendrogram type”
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4.10.2.6 Click on “OK” button to finish the calculations and the “Comparison”
window with the dendrogram will reappear
4.10.2.7 From the “Layout” drop-down menu, select the option “Show image”
4.10.2.8 From the “Composite” drop-down menu, select the option ”Show
quantification (values)” and the copy numbers will appear next to the
dendrogram
4.10.3 In order to create a minimum spanning tree:
4.10.3.1 Select the isolates to be included in the spanning tree
4.10.3.2 From the “Comparison” drop-down menu, select the option “Create
new comparison” and a “Comparison” window will appear
4.10.3.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics
versions lower than 5.0 or from the top left “Experiments” window in
BioNumerics 5.0.
4.10.3.4 From the “Clustering” drop-down menu, select the option
“Calculate…Minimum spanning tree (population modeling)” and a
“Minimum spanning tree” dialog box will appear
4.10.3.5 Make sure the default “Manhattan” is checked for “Coefficient” and
click “OK” and the “Minimum spanning tree” window with the tree
will appear.
4.10.3.6 You can find out the content for each node by clicking on them
individually.
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5. FLOW CHART:
6. BIBLIOGRAPHY:
7. CONTACTS:
7.1. Eija Hyytia-Trees, D.V.M., Ph.D.
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-3672
EHyytia-Trees@cdc.gov
7.2.Patti Lafon
PulseNet Methods Development and Reference Unit, EDLB, CDC
404-639-2828
PLafon@cdc.gov
7.3.Kara Cooper, Ph.D.
PulseNet Methods Development and Reference Unit, EDLB, CDC
404-639-4358
KCooper@cdc.gov
8. AMENDMENTS:
10/22/2009: the allele (= copy number) assignment will be based on a look-up table
listing the actual observed fragment size ranges instead of using the mathematical
formula that assigns alleles based on predicted fragment sizes. The look-up table
approach in allele assignment will facilitate the comparison of fragment size data from
the Beckman Coulter CEQ 8000 and the Applied Biosystems Genetic Analyzer 3130xl in
the same database.
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Appendix PND15-1
S. enterica serotype Typhimurium VNTR Allele List and Corresponding
Observed Fragment Sizes in the Beckman Coulter CEQ 8000 (BeckmanST look-up
table)
Note: This is posted on the CDC Team as a TXT-file so users may save it locally and use it with
the BioNumerics MLVA scripts. Every time the table is updated, a notification will be posted on
CDC Team together with the latest version of the table.
Count 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 ST3 164‐171 176‐181 187‐190 VERSION:
ST5 146‐149 151‐153 162‐164 168‐170 172‐176 179‐181 186‐188 191‐194 197‐199 203‐205 209‐210 214‐216 220‐222 226‐228 232‐233 238‐239 243‐245 249‐251 255‐256 261‐262 273‐274 278‐279 284‐286 290‐292 312‐314 ST7 114‐115 124‐126 130‐136 139‐145 151‐154 163‐164 172‐173 178‐182 187‐191 200‐201 218‐219 227‐229 232‐237 250‐256 282‐283 291‐293 321‐322 ST10 322‐323 335‐336 341‐343 347‐349 354‐356 360‐362 366‐368 373‐375 379‐381 385‐387 392‐394 398‐400 404‐406 411‐412 417‐418 424‐425 430‐431 437‐438 443‐446 450‐452 456‐458 463‐465 470‐471 476‐478 483‐484 489‐491 REPLACED BY:
ST2 249‐252 279‐281 289‐292 312‐316 319‐324 341‐343 351‐357 358‐369 387‐391 397‐407 425‐426 433‐435 ST6 202‐203 219‐221 225‐227 231‐233 234‐239 242‐245 247‐250 254‐257 259‐263 264‐268 272‐274 278‐280 284‐287 290‐292 296‐298 302‐303 305‐309 314‐315 320‐322 325‐327 331‐333 336‐338 343‐344 349‐350 355‐357 361‐362 367‐368 ST8 294‐296 327‐328 359‐360 373‐375 384‐385 399‐400 432‐435 448‐449 460‐464 471‐472 485‐486 495‐497 501‐508 512‐513 526‐531 AUTHORIZED BY:
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31 32 33 34 35 36 37 38 39 40 VERSION:
496‐498 509‐511 516‐517 523‐525 REPLACED BY:
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532‐537 547‐552 553‐565 580‐584 587‐592 599‐609 610‐618 622‐624 631‐636 AUTHORIZED BY:
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PULSENET STANDARD OPERATING PROCEDURE FOR
ANALYSIS OF MLVA DATA OF SHIGA TOXIN-PRODUCING
ESCHERICHIA COLI O157 (STEC O157) IN BIONUMERICSAPPLIED BIOSYSTEMS GENETIC ANALYZER DATA
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1. PURPOSE: to describe the standardized protocol for analysis of MLVA data of
Shiga toxin-producing Escherichia coli O157 (STEC O157) in BioNumerics.
2. SCOPE: to provide the PulseNet participants with a single protocol for analyzing
MLVA data of STEC O157, thus ensuring inter-laboratory comparability of the
generated results.
3. DEFINITIONS:
3.1 MLVA: Multiple-locus variable-number tandem repeat analysis
3.2 VNTR: Variable-number tandem repeat
3.3 CDC: Centers for Disease Control and Prevention
3.4 SOP: Standard Operating Procedure
4. RESPONSIBILITIES/PROCEDURE
4.1 Software needed for data analysis
4.1.1 BioNumerics version 3.0 or higher
4.1.2 Customized scripts for data import (VNTRImport_v4.bns), copy number
calculation (= allele assignment) (VNTRCalc_v4.bns) and troubleshooting
(VNTRReport.bns). An additional script (VNTRDetails_v2.bns) facilitates
special functions such as calculation of diversity indeces.
4.1.3 Look-up table (ABIEcoli.txt) for allele size ranges
4.2 General overview
4.2.1 The import process consists of three major steps:
4.2.1.1 Exporting the appropriate data file from the Genetic Analyzer system
(see laboratory protocol step 4.10.1.15)
4.2.1.2 Importing this file into BioNumerics (using the script
VNTRImport_v4.bns)
4.2.1.3 Determining the copy numbers (assigning alleles) for each VNTR
(using the script VNTRCalc_v4.bns and the look-up table ABIEcoli.txt)
4.2.1.3.1 VNTRCalc_v4.bns script facilitates the allele assignment in two
different ways:
4.2.1.3.1.1 “ABIEcoli” – assigns the copy number based on the lookup table (appendix PND16-1)
4.2.1.3.1.2 “Predicted” – calculates the copy number based on the
mathematical formula: (Observed fragment size –
offset)/repeat size (not an option for the ABI data)
4.3 Required import format
4.3.1 The data should be exported from the GeneMapper software as a .txt (tabdelimited) text file containing the fragment length information. Multiple
columns will be present in the file but only four columns are of interest:
“Dye/Sample Peak”, “Sample File Name”, “Size” and “Height”. All other
columns will be ignored by the script during import. The field “Sample File
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Name” should contain information designating the BioNumerics key
number (isolate identifier) loaded in each lane of the sequencer, as well as
which VNTR mastermix or reaction was loaded. The import script will
ignore any text that appears beyond the dot “.” in the “Sample File Name”
field.
4.4 Setting up a new database
4.4.1 Click “New” to set up a new database
4.4.2 Type in the name of your new database (for example O157MLVA) and
click ”Next”
4.4.3 Select the default directory to set up a new database and click “Next”
4.4.4 Choose “Yes” to enable creation of log files and click “Finish”
4.4.5 Choose the default “Use the local database” and click “OK”
4.4.6 Go to: Program files → BioNumerics → Data → O157MLVA and create
two folders named “Scripts” and “VNTRtables”.
4.4.7 Save the four MLVA specific scripts (VNTRImport_v4.bns,
VNTRCalc_v4.bns, VNTRReport.bns, VNTRDetails_v2.bns) in the “Scripts”
folder and the look-up table (ABIEcoli.txt) in the “VNTRtables” folder
4.5 Importing a peak file for the first time
4.5.1 Review the peak file: make sure that the ROX labelled molecular size
standard ran properly without skipping any peaks. Remove any data (failed
reactions, controls, internal ladder) that you don’t want to import in the
BioNumerics from the txt file. Re-name and re-save the txt file either on
your hard drive or on the flash drive.
4.5.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “ABI GeneMapper peak
file”. The script pops up a file dialog box, prompting for the name of the file
to import. Select the appropriate file and click “OK”. A second dialog box
pops up, prompting for a variety of other information:
4.5.2.1 Fingerprint file name. This is the name of the fingerprint file that will
be used in BioNumerics. Leave this unchanged, unless a file has
already been imported with the same name. In this case, change the file
name into a new, unique and informative name.
4.5.2.2 Dyes to import. The dye R (Rox) contains only reference markers and
does not need to be imported. Use this list to select what dyes will be
imported; they are B (FAM), G (HEX) and Y (Calred 590). Each dye
will be stored in a different file, resulting from appending the dye
name to the fingerprint file name.
4.5.2.3 Assign reference positions. Leave this option checked.
4.5.2.4 Select imported isolates. Leave this option checked.
4.5.2.5 Pool tags. For this protocol, there are two pool tags representing the
two PCR reactions for each isolate (all loaded on different wells) and
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4.5.2.7
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tagged with names “R1” and “R2 “. The two tags should appear in the
list. If they do not, they can be added by selecting “add” to update.
If everything is filled in appropriately, click “OK” to start the import.
NOTE: These settings are automatically saved and reloaded the next
time this script is run.
4.5.3 When the script is finished, it has:
4.5.3.1 Created a fingerprint type “VNTRFpr” with appropriate settings.
4.5.3.2 Created fingerprint types with appropriate settings for each dye and
each pool used. The names of these fingerprint types are
“VNTRFpr”+”pool name”+”dye” (for example: VNTRFprR1_Y).
4.5.3.3 Created fingerprint files for each dye in the fingerprint type
“VNTRFpr”, and imported the band information in these lanes. The
individual lanes are assigned to their appropriate pool fingerprint type,
according to the known pool tags found in the “Sample File Name”
field.
4.5.3.4 Created new database entries for all isolates found.
4.5.3.5 Linked the fingerprints to their corresponding entries.
4.5.3.6 Selected all isolates for which fingerprint data was imported.
4.6 Determining the copy numbers for the first time (allele assignment)
4.6.1 Run the script VNTRCalc_v4 from the scripts menu. The first time this script
is run, it will automatically create a new character set called “VNTR_vals”.
This character set will hold the copy numbers for each VNTR. The script
will also create a second character set called “VNTR_frags” which holds the
fragment size with a highest fluorescence for each VNTR. Click “OK” on
the two pop up windows notifying that these two character sets will be
created.
4.6.2 Before the script can determine copy numbers from the fragment length
information, it should have sufficient information about what VNTRs can
be found in which pool and dye, what are the repeat lengths, etc. To this
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end, the script pops up a dialog box called “VNTR assign”. This dialog box
contains a list of defined VNTRs that is initially empty. To enter the
parameters of the first VNTR, click on “Add”. This brings up a second
dialog box, prompting for all properties of this first VNTR. Refer to the
attachment PNL-4 in the laboratory protocol for all necessary information
to load the specifications for each VNTR.
4.6.3 The VNTR information includes:
4.6.3.1 Name. This name will be used for further reference and for the
character name in the character type VNTR_vals. Each VNTR should
have a unique name (for example VNTR_3, VNTR_34, etc).
4.6.3.2 Offset. Each fragment consists of a repeat portion and a constant
portion, due to the fact that the primers do not occur exactly at the start
and the end of the repeat region. This parameter specifies the size (in
base pairs) of the constant portion.
4.6.3.3 Repeat length. This parameter specifies the size of a unit repeat block
(in base pairs).
4.6.3.4 Copy range. These parameters specify the minimum and maximum
number of copies that the script will consider during the copy number
determination.
4.6.3.5 Tolerance. This specifies the maximum difference between the
expected fragment length (calculated by the software) and the actual
length estimated by the sequencer.
4.6.3.6 Take from fingerprint type. This drop-down list should be used to
indicate on what fingerprint type this VNTR was run. The fingerprint
type is determined by the dye and the pool tag.
4.6.4 If all parameters are filled in appropriately, click OK to add the VNTR and
return to the previous dialog box (“VNTR assign”). Repeat the same actions
to enter the information for all VNTRs used for this protocol. When all
VNTRs are entered, make sure the box for “Code absent as negative” is
checked. Select “ABIEcoli” from the “Fragment ranges” dropdown menu
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and press the “Save&Assign” button to let the script assign copy numbers
for the currently selected entries.
4.6.4.1 NOTE 1: pressing “Save&Quit” would store the information without
assigning copy numbers for the current selection.
4.6.4.2 NOTE 2: all information regarding the VNTRs is stored with the
database. The next time the script is run, the VNTR definitions will be
loaded automatically. New incoming VNTR data can be processed
simply by selecting the corresponding data entries, launching the script
VNTRCalc_v4 from the “Scripts” drop-down menu, and pressing the
button “Save&Assign”.
4.6.4.3 For every fragment with the highest fluorescence level in the
fingerprint type (= the combination of the reaction and the dye), the
script will assign an allele type (=a copy number) based on the
fragment size ranges specified in the “ABIEcoli” look-up table
(attachment PND16-1). Note that, within the same fingerprint type,
more than one VNTR can be loaded if there is no overlap within the
fragment size ranges.
4.6.5
When the script has completed, all copy numbers for all VNTRs for the
currently selected entries are determined.
4.6.5.1 NOTE: Two types of problems may arise during the process (if one or
more such errors were encountered during the calculations, an error
report is displayed listing all the problems):
4.6.5.1.1
None of the peaks present in a fingerprint are compatible with
the fragment size ranges in the look-up table. In this case, the
corresponding character value will be scored “-2.0”. In this
situation, run the “VNTRReport” script from the “Scripts” dropdown menu to verify whether the problem occurred because the
VNTR failed to amplify (mutations, insertions or deletions in the
primer annealing region) or because the VNTR had a fragment
size outside the acceptable range. In the latter case, rerun the
reaction to confirm the accuracy and reproducibility of the
sizing. The isolate should be submitted to CDC for confirmation
if the size upon retesting is still outside the fragment range
specified in the look-up table.
4.6.5.1.2
More than one peak in a fingerprint is compatible with an
acceptable fragment size range. In this case, the script will use
the solution that corresponds to the peak with the largest “peak
height” value.
4.7
Importing a peak file on a routine basis
4.7.1
Review the peak file: make sure that the ROX labelled molecular size
standard ran properly without skipping any peaks. Remove any data (failed
reactions, controls, internal ladder) that you don’t want to import in the
VERSION:
REPLACED BY:
AUTHORIZED BY:
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4.7.2
4.7.3
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BioNumerics from the txt file. Re-name and re-save the txt file either on
your hard drive or on the flash drive.
In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “ABI GeneMapper peak
file”. The script pops up a file dialog box, prompting for the name of the file
to import. Select the appropriate file and click “OK”. “VNTR/Import peak
data” dialog box will appear. De-select “R” from the “Dyes to import” and
click “OK”.
Run the script VNTRCalc_v4 from the scripts menu. “VNTR assign”
dialog box will appear. Select “ABIEcoli” from the “Fragment ranges”
dropdown menu and click “Save&Assign” to assign allele numbers.
4.8 Verifying the allele assignment
4.8.1 For each isolate in the database that has some VNTR data associated, you
can click on the “VNTR frags” and “VNTR vals” entries in the list to open
the “Entry edit” windows.
4.8.2
If the VNTRCalc_v4 script did not detect a fragment for a VNTR, refer to
the step 4.6.5.1.1 of this protocol for details on how to proceed. If the
fragment size is slightly outside the range specified in the “ABIEcoli” lookup table it is possible to manually assign a temporary allele size and type for
that VNTR until an official confirmation has been performed by CDC
(Note: only the CDC database managers are allowed to modify the
ABIEcoli.txt file. Once a modification has been made, the modified file will
be posted on CDCTeamSupport along with a notification that the file has
been modified). To manually assign an allele size and type click on the
fragment size in the “VNTRfrags” entry edit window or the allele type in
the “VNTR vals” entry edit window. This will open the “Change character
value” window in BioNumerics lower than 5.0. In BioNumerics 5.0 the
allele size or type can be directly highlighted and changed in the entry edit
window (Screen shots below)
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4.9 Visualizing the data
4.9.1 In order to display copy numbers next to a dendrogram in a comparison, first
create a “composite data set” that holds the VNTR data.
4.9.1.1 From the “Experiments” drop-down menu, select the option “Create new
composite data set…”, enter a name (e.g. VNTR_cmp), and click the
“OK” button. The “Composite data set ‘VNTR_cmp’” window will
appear.
4.9.1.2 Highlight the experiment VNTR_vals and from the “Experiment” dropdown menu, select the option “Use in composite data set”. Close the
window.
4.9.2 Next time a comparison window is opened (see below “Analysis of the
VNTR data”), there will be a new experiment VNTR_cmp listed in the
bottom of the window (BioNumerics versions lower than 5.0) or in the top
left corner of the window (BioNumerics version 5.0). This experiment will
facilitate the display of a spreadsheet-like view of the copy numbers (note
that it may be necessary to scroll the experiment list to the right with the
arrow button to bring VNTR_cmp in display in BioNumerics versions lower
than 5.0). This can be shown next to a dendrogram analysis of the data set.
4.10 Analysis of the VNTR data
4.10.1. The VNTR data contained in the character set VNTR_vals can be analysed
in BioNumerics with all the tools that are available to character data. That
includes cluster analysis with a variety of methods and similarity
coefficients. For VNTR data, the coefficients that make most sense are:
4.10.1.1 Categorical: preferred if differences in copy numbers should be
treated in a qualitative way. This is the only option for creating
dendrograms using MLVA data.
4.10.1.2 Manhattan: preferred if differences in copy numbers should be treated
in a quantitative way (larger difference means more distantly related
organisms). This coefficient can be used to construct minimum
spanning trees.
4.10.2 In order to create a dendrogram:
4.10.2.1 Select the isolates to be included in the dendrogram
4.10.2.2 From the “Comparison” drop-down menu, select the option “Create
new comparison” and a “Comparison” window will appear
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4.10.2.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics
versions lower than 5.0 or from the top left “Experiments” window in
BioNumerics 5.0.
4.10.2.4 From the “Clustering” drop-down menu, select the option
“Calculate…Cluster analysis (similarity matrix)” and a “Composite
data set comparison” dialog box will appear
4.10.2.5 Select “Categorical” for “Multi-state coefficient” and “UPGMA” for
“Dendrogram type”
4.10.2.6 Click on “OK” button to finish the calculations and the “Comparison”
window with the dendrogram will reappear
4.10.2.7 From the “Layout” drop-down menu, select the option “Show image”
4.10.2.8 From the “Composite” drop-down menu, select the option ”Show
quantification (values)” and the copy numbers will appear next to the
dendrogram
4.10.3 In order to create a minimum spanning tree:
4.10.3.1 Select the isolates to be included in the spanning tree
4.10.3.2 From the “Comparison” drop-down menu, select the option “Create
new comparison” and a “Comparison” window will appear
4.10.3.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics
versions lower than 5.0 or from the top left “Experiments” window in
BioNumerics 5.0.
4.10.3.4 From the “Clustering” drop-down menu, select the option
“Calculate…Minimum spanning tree (population modeling)” and a
“Minimum spanning tree” dialog box will appear
4.10.3.5 Make sure the default “Manhattan” is checked for “Coefficient” and
click “OK” and the “Minimum spanning tree” window with the tree
will appear.
4.10.3.6 You can find out the content for each node by clicking on them
individually.
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5.
FLOW CHART:
6.
BIBLIOGRAPHY:
7.
CONTACTS:
7.1 Eija Hyytia-Trees, D.V.M., Ph.D.
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-3672
EHyytia-Trees@cdc.gov
7.2 Patti Lafon
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-2828
PLafon@cdc.gov
7.3 Kara Cooper, Ph.D.
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-4358
KCooper@cdc.gov
8.
AMENDMENTS:
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Appendix PND16-1
Fragment ranges for the STEC O157 VNTR alleles (ABIEoli look-up table)
Count
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
VNTR_3
339-340
343-346
349-352
355-358
361-364
367-370
373-376
380-382
385-388
391-393
397-401
404-406
410-411
416-417
422-424
429-430
434-435
440-441
447-448
453-455
459-461
466-467
472-473
VNTR_34
169-171
187-188
205-206
223-224
242-243
260-262
278-280
295-298
VNTR_9
474-475
485-486
491-492
496-498
502-504
508-510
514-516
520-522
526-528
531-534
536-539
543-545
549-551
555-557
560-563
566-569
572-575
578-581
584-585
590-591
596-597
601-603
607-608
613-614
VNTR_25
122-124
127-128
132-134
138-140
144-146
151-152
157-158
162-164
168-169
174-175
191-192
VNTR_17
VNTR_19
135-136
140-142
147-148
153-155
159-161
166-167
172-173
177-179
184-185
190-191
196-197
201-202
283-284
291-292
297-298
302-304
309-310
315-316
321-322
327-328
332-334
338-340
344-345
VNTR_36
123-124
129-131
136-137
141-144
150-151
157-158
164-165
170-172
177-179
184-185
191-192
198-199
204-205
REPLACED BY:
163-165
169-171
175-176
181-182
187-188
193-194
199-200
205-206
211-212
217-218
223-224
229-231
236-237
241-244
227-228
273-274
Note: This document is posted as a separate .txt file on CDC Team
VERSION:
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1. PURPOSE: to describe the standardized protocol for analysis of MLVA data of
Salmonella enterica serotype Typhimurium in BioNumerics.
2. SCOPE: to provide the PulseNet participants with a single protocol for analyzing
MLVA data of S. enterica serotype Typhimurium, thus ensuring inter-laboratory
comparability of the generated results.
3. DEFINITIONS:
3.1 MLVA: Multiple-locus variable-number tandem repeat analysis
3.2 VNTR: Variable-number tandem repeat
3.3 CDC: Centers for Disease Control and Prevention
3.4 SOP: Standard Operating Procedure
4. RESPONSIBILITIES/PROCEDURE
4.1 Software needed for data analysis
4.1.1 BioNumerics version 3.0 or higher
4.1.2 Customized scripts for data import (VNTRImport_v4.bns), copy number
calculation (= allele assignment) (VNTRCalc_v4.bns), troubleshooting
(VNTRReport.bns), and support functions (VNTRDetails_v2.bns)
4.1.3 Look-up table (ABIST.txt) for allele size ranges
4.2 General overview
4.2.1 The import process consists of three major steps:
4.2.1.1
Exporting the appropriate data file from the Genetic Analyzer system
(see laboratory protocol PNL24 step 4.10.1.15)
4.2.1.2
Importing this file into BioNumerics (using the script
VNTRImport_v4.bns)
4.2.1.3
Determining the copy numbers (assigning alleles) for each VNTR
(using the script VNTRCalc_v4.bns and the look-up table ABIST.txt)
4.2.1.3.1 VNTRCalc_v4.bns script facilitates the allele assignment in two
different ways:
4.2.1.3.1.1 “ABIST” – assigns the copy number based on the look-up
table (PND17-1)
4.2.1.3.1.2 “Predicted” – calculates the copy number based on the
mathematical formula: (Observed fragment size –
offset)/repeat size (not an option for the ABI data)
4.3 Required import format
4.3.1 The data should be exported from the GeneMapper software as a .txt (tabdelimited) text file containing the fragment length information. Multiple
columns will be present in the file but only four columns are of interest:
“Dye/Sample Peak”, “Sample File Name”, “Size” and “Height”. All other
columns will be ignored by the script during import. The field “Sample File
Name” should contain information designating the BioNumerics key
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number (isolate identifier) loaded in each lane of the sequencer, as well as
which VNTR mastermix or reaction was loaded. The import script will
ignore any text that appears beyond the dot “.” in the “Sample File Name”
field.
4.3.1.1
NOTE: no spaces or underscores are allowed between the strain ID
and the reaction ID or between the reaction ID and the dot “.” in the
RN field.
4.4 Setting up a new database
4.4.1 Click “New” to set up a new database
4.4.2 Type in the name of your new database (for example STMLVA) and click
”Next”
4.4.3 Select the default directory to set up a new database and click “Next”
4.4.4 Choose “Yes” to enable creation of log files and click “Finish”
4.4.5 Choose the default “Use the local database” and click “OK”
4.4.6 Go to: Program files → BioNumerics → Data → STMLVA and create two
folders named “Scripts” and “VNTRtables”.
4.4.7 Save the four MLVA specific scripts (VNTRImport_v4.bns,
VNTRCalc_v4.bns, VNTRReport.bns, VNTRDetails_v2.bns) in the “Scripts”
folder and the look-up table (ABIST.txt) in the “VNTRtables” folder
4.5 Importing a peak file for the first time
4.5.1 Review the peak file: make sure that the ROX labelled molecular size
standard ran properly without skipping any peaks. Remove any data (failed
reactions, controls, internal ladder) that you don’t want to import in the
BioNumerics from the txt file. Re-name and re-save the txt file either on
your hard drive or on the flash drive.
4.5.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “ABI GeneMapper peak
file”. The script pops up a file dialog box, prompting for the name of the file
to import. Select the appropriate file and click “OK”. A second dialog box
pops up, prompting for a variety of other information:
4.5.2.1 Fingerprint file name. This is the name of the fingerprint file that
will be used in BioNumerics. Leave this unchanged, unless a file has
already been imported with the same name. In this case, change the
file name into a new, unique and informative name.
4.5.2.2 Dyes to import. The dye R (Rox) contains only reference markers
and does not need to be imported. Use this list to select what dyes
will be imported; they are B (FAM), G (HEX) and Y (Calred 590).
Each dye will be stored in a different file, resulting from appending
the dye name to the fingerprint file name.
4.5.2.3 Assign reference positions. Leave this option checked.
4.5.2.4 Select imported isolates. Leave this option checked.
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4.5.2.5
4.5.2.6
4.5.2.7
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Pool tags. For this protocol, there are two pool tags representing the
two PCR reactions for each isolate (all loaded on different wells) and
tagged with names “R1” and “R2 “. The two tags should appear in the
list. If they do not, they can be added by selecting “add” to update.
If everything is filled in appropriately, click “OK” to start the import.
NOTE: These settings are automatically saved and reloaded the next
time this script is run.
4.5.3 When the script is finished, it has:
4.5.3.1
Created a fingerprint type “VNTRFpr” with appropriate settings.
4.5.3.2
Created fingerprint types with appropriate settings for each dye and
each pool used. The names of these fingerprint types are
“VNTRFpr”+”pool name”+”dye” (for example: VNTRFprR1_Y).
4.5.3.3
Created fingerprint files for each dye in the fingerprint type
“VNTRFpr”, and imported the band information in these lanes. The
individual lanes are assigned to their appropriate pool fingerprint type,
according to the known pool tags found in the “Sample File Name”
field.
4.5.3.4
Created new database entries for all isolates found.
4.5.3.5
Linked the fingerprints to their corresponding entries.
4.5.3.6
Selected all isolates for which fingerprint data was imported.
4.6 Determining the copy numbers for the first time (allele assignment)
4.6.1 Run the script VNTRCalc_v4 from the scripts menu. The first time this script
is run, it will automatically create a new character set called “VNTR_vals”.
This character set will hold the copy numbers for each VNTR. The script
will also create a second character set called “VNTR_frags” which holds the
fragment size with a highest fluorescence for each VNTR. Click “OK” on
the two pop up windows notifying that these two character sets will be
created.
4.6.2 Before the script can determine copy numbers from the fragment length
information, it should have sufficient information about what VNTRs can
be found in which pool and dye, what are the repeat lengths, etc. To this
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end, the script pops up a dialog box called “VNTR assign”. This dialog box
contains a list of defined VNTRs that is initially empty. To enter the
parameters of the first VNTR, click on “Add”. This brings up a second
dialog box, “Edit VNTR”, prompting for all properties of this first VNTR.
Refer to the attachment PNL24-4 in the laboratory protocol for all necessary
information to load the specifications for each VNTR.
4.6.3 The VNTR information includes:
4.6.3.1 Name. This name will be used for further reference and for the
character name in the character type VNTR_vals. Each VNTR should
have a unique name (for example ST3, ST5, etc).
4.6.3.2 Offset. Each fragment consists of a repeat portion and a constant
portion, due to the fact that the primers do not occur exactly at the start
and the end of the repeat region. This parameter specifies the size (in
base pairs) of the constant portion.
4.6.3.3 Repeat length. This parameter specifies the size of a unit repeat block
(in base pairs).
4.6.3.4 Copy range. These parameters specify the minimum and maximum
number of copies that the script will consider during the copy number
determination.
4.6.3.5 Tolerance. This specifies the maximum difference between the
expected fragment length (calculated by the software) and the actual
length estimated by the sequencer.
4.6.3.6 Take from fingerprint type. This drop-down list should be used to
indicate on what fingerprint type this VNTR was run. The fingerprint
type is determined by the dye and the pool tag.
4.6.4 If all parameters are filled in appropriately, click OK to add the VNTR and
return to the previous dialog box (“VNTR assign”). Repeat the same actions
to enter the information for all VNTRs used for this protocol. When all
VNTRs are entered, make sure the box for “Code absent as negative” is
checked. Select “ABIST” from the “Fragment ranges” dropdown menu and
press the “Save&Assign” button to let the script assign copy numbers for
the currently selected entries.
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4.6.4.1
NOTE 1: pressing “Save&Quit” would store the information without
assigning copy numbers for the current selection.
4.6.4.2 NOTE 2: all information regarding the VNTRs is stored with the
database. The next time the script is run, the VNTR definitions will be
loaded automatically. New incoming VNTR data can be processed
simply by selecting the corresponding data entries, launching the script
VNTRCalc_v4 from the “Scripts” drop-down menu, and pressing the
button “Save&Assign”.
4.6.4.3 For every fragment with the highest fluorescence level in the
fingerprint type (= the combination of the reaction and the dye), the
script will assign an allele type (=a copy number) based on the
fragment size ranges specified in the “ABIST” look-up table
(attachment PND17-1). Note that, within the same fingerprint type,
more than one VNTR can be loaded if there is no overlap within the
fragment size ranges.
4.6.5
When the script has completed, all copy numbers for all VNTRs for the
currently selected entries are determined.
4.6.5.1 NOTE: Two types of problems may arise during the process (if one or
more such errors were encountered during the calculations, an error
report is displayed listing all the problems):
4.6.5.1.1
None of the peaks present in a fingerprint are compatible with
the fragment size ranges in the look-up table. In this case, the
corresponding character value will be scored “-2.0”. In this
situation, run the “VNTRReport” script from the “Scripts” dropdown menu to verify whether the problem occurred because the
VNTR failed to amplify (mutations, insertions or deletions in the
primer annealing region or the whole VNTR array and flanking
sequences missing) or because the VNTR had a fragment size
outside the acceptable range. In the latter case, rerun the reaction
to confirm the accuracy and reproducibility of the sizing. The
isolate should be submitted to CDC for confirmation if the size
upon retesting is still outside the fragment range specified in the
look-up table.
4.6.5.1.2
More than one peak in a fingerprint is compatible with an
acceptable fragment size range. In this case, the script will use
the solution that corresponds to the peak with the largest “peak
height” value.
4.7
Importing a peak file on a routine basis
4.7.1
Review the peak file: make sure that the ROX labelled molecular size
standard ran properly without skipping any peaks. Remove any data (failed
reactions, controls, internal ladder) that you don’t want to import in the
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BioNumerics from the txt file. Re-name and re-save the txt file either on
your hard drive or on the flash drive.
4.7.2
In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “ABI GeneMapper peak
file”. The script pops up a file dialog box, prompting for the name of the file
to import. Select the appropriate file and click “OK”. “VNTR/Import peak
data” dialog box will appear. De-select “R” from the “Dyes to import” and
click “OK”.
4.7.3
Run the script VNTRCalc_v4 from the scripts menu. “VNTR assign”
dialog box will appear. Select “ABIST” from the “Fragment ranges”
dropdown menu and click “Save&Assign” to assign allele numbers.
4.8 Verifying the allele assignment
4.8.1 For each isolate in the database that has some VNTR data associated, you
can click on the “VNTR frags” and “VNTR vals” entries in the list to open
the “Entry edit” windows.
4.8.2
If the VNTRCalc_v4 script did not detect a fragment for a VNTR, refer to
the step 4.6.5.1.1 of this protocol for details on how to proceed. If the
fragment size is slightly outside the range specified in the “ABIST” look-up
table it is possible to manually assign a temporary allele size and type for
that VNTR until an official confirmation has been performed by CDC
(Note: only the CDC PulseNet database managers are allowed to modify
the ABIST.txt file. Once a modification has been made, the modified file
will be posted on CDCTeamSupport along with a notification that the file
has been modified). To manually assign an allele size and type click on the
fragment size in the “VNTRfrags” entry edit window or the allele type in
the “VNTR vals” entry edit window. This will open the “Change character
value” window in BioNumerics lower than 5.0. In BioNumerics 5.0, the
allele size or type can be directly highlighted and changed in the entry edit
window (Screen shots below)
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4.9 Visualizing the data
4.9.1 In order to display copy numbers next to a dendrogram in a comparison, first
create a “composite data set” that holds the VNTR data.
4.9.1.1 From the “Experiments” drop-down menu, select the option “Create new
composite data set…”, enter a name (e.g. VNTR_cmp), and click the
“OK” button. The “Composite data set VNTR_cmp” window will
appear.
4.9.1.2 Highlight the experiment VNTR_vals and from the “Experiment” dropdown menu, select the option “Use in composite data set”. Close the
window.
4.9.2 Next time a comparison window is opened (see below “Analysis of the
VNTR data”), there will be a new experiment VNTR_cmp listed in the
bottom of the window (BioNumerics versions lower than 5.0) or in the top
left corner of the window (BioNumerics version 5.0). This experiment will
facilitate the display of a spreadsheet-like view of the copy numbers that
can be shown next to a dendrogram analysis of the data set.
4.9.2.1 NOTE: it may be necessary to scroll the experiment list to the right with
the arrow button to bring VNTR_cmp in display in BioNumerics
versions lower than 5.0).
4.10 Analysis of the VNTR data
4.10.1. The VNTR data contained in the character set VNTR_vals can be analysed
in BioNumerics with all the tools that are available to character data. That
includes cluster analysis with a variety of methods and similarity
coefficients. For VNTR data, the coefficients that make most sense are:
4.10.1.1 Categorical: preferred if differences in copy numbers should be
treated in a qualitative way. This is the only option for creating
dendrograms using MLVA data.
4.10.1.2 Manhattan: preferred if differences in copy numbers should be treated
in a quantitative way (larger difference means more distantly related
organisms). This coefficient can be used to construct minimum
spanning trees.
4.10.2 In order to create a dendrogram:
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4.10.2.1 Select the isolates to be included in the dendrogram
4.10.2.2 From the “Comparison” drop-down menu, select the option “Create
new comparison” and a “Comparison” window will appear
4.10.2.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics
versions lower than 5.0 or from the top left “Experiments” window in
BioNumerics 5.0.
4.10.2.4 From the “Clustering” drop-down menu, select the option
“Calculate…Cluster analysis (similarity matrix)” and a “Composite
data set comparison” dialog box will appear
4.10.2.5 Select “Categorical” for “Multi-state coefficient” and “UPGMA” for
“Dendrogram type”
4.10.2.6 Click on “OK” button to finish the calculations and the “Comparison”
window with the dendrogram will reappear
4.10.2.7 From the “Layout” drop-down menu, select the option “Show image”
4.10.2.8 From the “Composite” drop-down menu, select the option ”Show
quantification (values)” and the copy numbers will appear next to the
dendrogram
4.10.3 In order to create a minimum spanning tree:
4.10.3.1 Select the isolates to be included in the spanning tree
4.10.3.2 From the “Comparison” drop-down menu, select the option “Create
new comparison” and a “Comparison” window will appear
4.10.3.3 Select “VNTR_cmp” from the bottom of the window in BioNumerics
versions lower than 5.0 or from the top left “Experiments” window in
BioNumerics 5.0.
4.10.3.4 From the “Clustering” drop-down menu, select the option
“Calculate…Minimum spanning tree (population modeling)” and a
“Minimum spanning tree” dialog box will appear
4.10.3.5 Make sure the default “Manhattan” is checked for “Coefficient” and
click “OK” and the “Minimum spanning tree” window with the tree
will appear.
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CODE:
PND17
PULSENET STANDARD OPERATING PROCEDURE FOR
ANALYSIS OF MLVA DATA OF SALMONELLA ENTERICA
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BIOSYSTEMS GENETIC ANALYZER DATA
Effective Date:
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09
4.10.3.6 You can find out the content for each node by clicking on them
individually.
5.
FLOW CHART:
6.
BIBLIOGRAPHY:
7.
CONTACTS:
7.1 Eija Hyytia-Trees, D.V.M., Ph.D.
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-3672
EHyytia-Trees@cdc.gov
7.2 Patti Lafon
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-2828
PLafon@cdc.gov
7.3 Kara Cooper, Ph.D.
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-4358
KCooper@cdc.gov
8.
AMENDMENTS:
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AUTHORIZED BY:
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Appendix PND17-1
S. enterica serotype Typhimurium VNTR Allele List and Corresponding Observed
Fragment Sizes in the Applied Biosystems Genetic Analyzer 3130xl
(ABIST look-up table)
Note: This is posted on the CDCTeam as a TXT-file so users may save it locally and use it with
the BioNumerics MLVA scripts. Every time the table is updated, a notification will be posted on
CDCTeam together with the latest version of the table.
Count 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 ST3 177‐180 188‐192 200‐202 VERSION:
ST5 146‐147 149‐153 160‐164 166‐168 172‐174 178‐179 184‐185 190‐191 195‐197 201‐203 207‐208 213‐214 219‐220 225‐226 231‐233 237‐239 244‐245 249‐251 256‐257 261‐263 273‐275 280‐281 286‐287 292‐293 315‐316 ST7 121‐122 128‐130 137‐139 147‐151 157‐158 166‐167 175‐176 184‐185 193‐194 202‐203 220‐221 229‐230 239‐240 258‐259 286‐287 295‐296 321‐322 ST10 322‐323 340‐341 345‐347 351‐354 358‐359 364‐366 370‐372 376‐378 383‐384 389‐390 394‐396 401‐402 407‐409 413‐415 420‐421 424‐427 431‐433 438‐439 445‐446 451‐453 458‐460 464‐466 470‐472 477‐478 483‐484 REPLACED BY:
ST2 256‐257 286‐287 298‐299 324‐325 327‐334 351‐352 362‐366 370‐381 399‐401 409‐410 437‐439 448‐449 ST6 205‐206 222‐226 229‐230 235‐236 241‐243 247‐250 253‐255 259‐261 265‐267 271‐273 277‐279 283‐285 289‐290 295‐296 300‐302 306‐308 312‐314 318‐320 325‐326 330‐332 336‐337 342‐343 348‐349 354‐355 360‐361 366‐367 372‐373 ST8 298‐299 331‐332 364‐365 377‐378 391‐392 403‐404 436‐437 452‐454 464‐466 473‐474 491‐492 497‐499 503‐504 517‐518 AUTHORIZED BY:
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30 31 32 33 34 35 36 37 38 39 40 VERSION:
489‐491 495‐496 507‐508 514‐517 520‐521 REPLACED BY:
CODE:
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530‐532 537‐538 550‐552 556‐565 582‐584 585‐591 609‐610 616‐617 622‐623 AUTHORIZED BY:
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PULSENET STANDARD OPERATING PROCEDURE FOR
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SEROTYPE ENTERITIDIS IN BIONUMERICS-APPLIED
BIOSYSTEMS GENETIC ANALYZER DATA
CODE:
PND18
Effective Date:
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1. PURPOSE: to describe the standardized protocol for analysis of MLVA data of
Salmonella enterica serotype Enteritidis (S. enterica serotype Enteritidis) in
BioNumerics.
2. SCOPE: to provide the PulseNet participants with a single protocol for analyzing
MLVA data of S.enterica serotype Enteritidis, thus ensuring inter-laboratory
comparability of the generated results.
3. DEFINITIONS:
3.1 MLVA: Multiple-locus variable-number tandem repeat analysis
3.2 VNTR: Variable-number tandem repeat
3.3 CDC: Centers for Disease Control and Prevention
3.4 SOP: Standard operating procedure
4. RESPONSIBILITIES/PROCEDURE
4.1.
Software needed for data analysis
4.1.1. BioNumerics version 4.5 or higher
4.1.2. Customized scripts for data import (VNTRImport_v4.bns) and copy
number calculation (= allele assignment) (VNTRCalc_v4.bns),
troubleshooting (VNTRReport.bns), and support functions
(VNTRDetails_v2.bns)
4.1.3 Look-up table (ABISE.txt) for allele size ranges
4.2.
General overview
4.2.1. The import process consists of three major steps:
4.2.1.1.Exporting the appropriate data file from the Genetic Analyzer system
(see lab protocol PNL26 step 4.10.1.15)
4.2.1.2.Importing this file into BioNumerics (using the script
VNTRImport_v4.bns)
4.2.1.3.Determining the copy numbers (assigning alleles) for each VNTR
(using the script VNTRCalc_v4.bns and the look-up table ABISE.txt)
4.2.1.3.1. VNTRCalc_v4.bns script facilitates the allele assignment in two
different ways:
4.2.1.3.1.1. “ABISE” – assigns the copy number based on the look-up
table (PND18-1)
4.2.1.3.1.2.“Predicted” – calculates the copy number based on the
mathematical formula: (Observed fragment size –
offset)/repeat size (not an option for the ABI data)
4.3.
Required import format
4.3.1. The data should be exported from the GeneMapper software as a txt file
(tab-delimited text file) containing the fragment length information.
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Multiple columns will be present in the file but only four columns are of
interest: “Dye/Sample Peak”, “Sample File Name”, “Size” and “Height”.
All other columns will be ignored by the script during import. The field
“Sample File Name” should contain information designating the
BioNumerics key number (isolate identifier) loaded in each lane of the
sequencer, as well as which VNTR mastermix (reaction) was loaded. The
import script will ignore any text that appears beyond the dot “.” in the
“Sample File Name” field.
4.3.1.1. NOTE: no spaces or underscores are allowed between the strain ID
and the reaction ID or between the reaction ID and the dot “.” in the
RN field.
4.4.
Setting up a new database
4.4.1. Click “New” to set up a new database
4.4.2. Type in the name of your new database (for example SEMLVA) and click
“Next”
4.4.3. Select the default directory to set up a new database and click “Next”
4.4.4. Choose “Yes” to enable creation of log files and click “Finish”
4.4.5. Choose the default “Use the local database” and click “OK”
4.4.6. Go to: Program files → BioNumerics → Data → SEMLVA and create
two folders named “Scripts” and “VNTRtables”.
4.4.7. Save the four MLVA specific scripts (VNTRImport_v4.bns,
VNTRCalc_v4.bns, VNTRReport.bns, VNTRDetails_v2.bns) in the
“Scripts” folder and the look-up table (ABISE.txt) in the “VNTRtables”
folder
4.5.
Importing a peak file for the first time
4.5.1. Review the peak file: make sure that the ROX labelled molecular size
standard ran properly without skipping any peaks. Remove any data
(failed reactions, controls, internal ladder) that you don’t want to import in
the BioNumerics from the txt file. Re-name and re-save the txt file either
on your hard drive or on the flash drive.
4.5.2. In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “ABI GeneMapper peak
file”. The script pops up a file dialog box, prompting for the name of the
file to be imported. Select the appropriate file and click “OK”. A second
dialog box pops up, prompting for a variety of other information:
4.5.2.1. Fingerprint file name. This is the name of the fingerprint file that will
be used in BioNumerics. Leave this unchanged, unless a file has
already been imported with the same name. In this case, change the
file name into a new, unique and informative name.
4.5.2.2. Dyes to import. The dye R (Rox) contains only reference markers and
does not need to be imported. Use this list to select what dyes will be
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imported; they are B (FAM), G (HEX) and Y (Calred 590). Each dye
will be stored in a different file, resulting from appending the dye
name to the fingerprint file name.
4.5.2.3. Assign reference positions. Leave this option checked.
4.5.2.4. Select imported isolates. Leave this option checked.
4.5.2.5. Pool tags. For this protocol, there are two pool tags representing the
two PCR reactions for each isolate (each loaded on different wells)
and tagged with names “R1” and “R2 “. The two tags should appear in
the list. If they do not, they can be added by selecting “add” to update.
4.5.2.6. If everything is filled in appropriately, click “OK” to start the import.
4.5.2.7. NOTE: These settings are automatically saved and reloaded the next
time this script is run.
4.5.3. When the script is finished, it has:
4.5.3.1.Created a fingerprint type “VNTRFpr” with appropriate settings.
4.5.3.2.Created fingerprint types with appropriate settings for each dye and
each pool used. The names of these fingerprint types are
“VNTRFpr”+”pool name”+”dye” (for example: VNTRFprR1_Y).
4.5.3.3.Created fingerprint files for each dye in the fingerprint type
“VNTRFpr”, and imported the band information in these lanes. The
individual lanes are assigned to their appropriate pool fingerprint type,
according to the known pool tags found in the “Sample File Name”
field.
4.5.3.4.Created new database entries for all isolates found.
4.5.3.5.Linked the fingerprints to their corresponding entries.
4.5.3.6.Selected all isolates for which fingerprint data was imported.
4.6 Determining the copy numbers for the first time (allele assignment)
4.6.1 Run the script VNTRCalc_v4 from the scripts menu. The first time this
script is run, it will automatically create a new character set called
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“VNTR_vals”. This character set will hold the copy numbers for each
VNTR. The script will also create a second character set called
“VNTR_frags” which holds the fragment size with a highest fluorescence
for each VNTR. Click “OK” on the two pop-up windows notifying that
these two character sets will be created.
4.6.2 Before the script can determine copy numbers from the fragment length
information, it should have sufficient information about what VNTRs can
be found in which pool and dye, what are the repeat lengths, etc. To this
end, the script pops up a dialog box called “VNTR assign”. This dialog
box contains a list of defined VNTRs that is initially empty. To enter the
parameters of the first VNTR, click on “Add”. This brings up a second
dialog box, “Edit VNTR”, prompting for all properties of this first VNTR.
Refer to the attachment PNL26-4 in the laboratory protocol for all
necessary information to load the specifications for each VNTR.
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4.6.3 The VNTR information includes:
4.6.3.1 Name. This name will be used for further reference and for the
character name in the character type VNTR_vals. Each VNTR should
have a unique name (for example VNTR_1, VNTR_2, etc).
4.6.3.2 Offset. Each fragment consists of a repeat portion and a constant
portion, due to the fact that the primers do not locate exactly at the
start and the end of the repeat region. This parameter specifies the size
(in base pairs) of the constant portion.
4.6.3.3 Repeat length. This parameter specifies the size of the repeat block
unit (in base pairs).
4.6.3.4 Copy range. These parameters specify the minimum and maximum
number of copies that the script will consider during the copy number
determination.
4.6.3.5 Tolerance. This specifies the maximum difference between the
expected fragment length (calculated by the software) and the actual
length estimated by the sequencer.
4.6.3.6 Take from fingerprint type. This drop-down list should be used to
indicate on what fingerprint type this VNTR was run. The fingerprint
type is determined by the dye and the pool tag.
4.6.4. If all parameters are filled in appropriately, click OK to add the VNTR and
return to the previous dialog box (“VNTR assign”). Repeat the same
actions to enter the information for all VNTRs used for this protocol.
When all VNTRs are entered, make sure the box for “Code absent as
negative” is checked. Select “ABISE” from the “Fragment ranges”
dropdown menu and press the “Save &Assign” button to let the script
assign copy numbers for the currently selected entries.
4.6.4.1. NOTE 1: pressing “Save&OK” would store the information without
assigning copy numbers for the current selection.
4.6.4.2. NOTE 2: all information regarding the VNTRs is stored with the
database. The next time the script is run, the VNTR definitions will be
loaded automatically. New incoming VNTR data can be processed
simply by selecting the corresponding data entries, launching the script
VNTRCalc_v4 from the “Scripts” drop-down menu, and pressing the
button “Save&Assign”.
4.6.4.3.For every fragment with the highest fluorescence level in the
fingerprint type (= the combination of the reaction and the dye), the
script will assign an allele type (=a copy number) based on the
fragment size ranges specified in the “ABISE” look-up table
(Appendix PND18-1). Note that, within the same fingerprint type,
more than one VNTR can be loaded if there is no overlap within the
fragment size ranges.
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4.6.5. When the script has completed, copy numbers for all VNTRs for the
currently selected entries are determined.
4.6.5.1. NOTE: Two types of problems may arise during the process (if one
or more such errors were encountered during the calculations, an error
report is displayed listing all the problems):
4.6.5.1.1. None of the peaks present in a fingerprint are compatible with
the fragment size ranges in the look-up table. In this case, the
corresponding character value will be scored “-2.0”. In this
situation, run the “VNTRReport” script from the “Scripts”
drop-down menu to verify whether the problem occurred
because the VNTR failed to amplify (mutations, insertions or
deletions in the primer annealing region, the whole VNTR
array and flanking sequences missing, VNTR located on a
plasmid) or because the VNTR had a fragment size outside the
acceptable range (incomplete repeats, new alleles). In the latter
case, rerun the reaction to confirm the accuracy and
reproducibility of the sizing. The isolate should be submitted to
CDC for confirmation if the size upon retesting is still outside
the fragment ranges specified in the look-up table.
4.6.5.1.2. More than one peak in a fingerprint is compatible with an
acceptable fragment size range. In this case, the script will use
the solution that corresponds to the peak with the largest “peak
height” value.
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CODE:
PND18
Effective Date:
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4.7.Importing a peak file and assigning alleles on a routine basis
4.7.1 Review the peak file: make sure that the ROX labelled molecular size
standard ran properly without skipping any peaks. Remove any data
(failed reactions, controls, internal ladder) that you don’t want to import in
the BioNumerics from the txt file. Re-name and re-save the txt file either
on your hard drive or on the flash drive.
4.7.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “ABI GeneMapper peak
file”. The script pops up a file dialog box, prompting for the name of the
file to be imported. Select the appropriate file and click “OK”.
“VNTR/Import peak data” dialog box will appear. De-select “R” from the
“Dyes to import” and click “OK”.
4.7.3 Run the script VNTRCalc_v4 from the scripts menu. “VNTR assign”
dialog box will appear. Select “ABISE” from the “Fragment ranges” dropdown menu and click “Save&Assign” to assign allele numbers.
4.8 Verifying the accuracy of the allele types
4.8.1 For each isolate in the database that has some VNTR data associated, you
can click on the “VNTR frags” and “VNTR vals” entries in the list to open
the “Entry edit” windows.
4.8.2
If the VNTRCalc_v4 script did not detect a fragment for a VNTR, refer to
the step 4.6.5.1.1 of this protocol for details on how to proceed. If the
fragment size is slightly outside the range specified in the “ABISE” lookup table it is possible to manually assign a temporary allele size and type
for that VNTR until an official confirmation has been performed by CDC
(Note: only the CDC PulseNet database managers are allowed to modify
the ABISE.txt file. Once a modification has been made, the modified file
will be posted on CDCTeamSupport along with a notification that the file
has been modified). To manually assign an allele size and type click on the
fragment size in the “VNTRfrags” entry edit window or the allele type in
the “VNTR vals” entry edit window. This will open the “Change character
value” window in BioNumerics lower than 5.0. In BioNumerics 5.0, the
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allele size or type can be directly highlighted and changed in the entry edit
window (Screen shots below)
4.9. Visualizing the data
4.9.1. In order to display copy numbers next to a dendrogram in a comparison,
first create a “composite data set” that holds the VNTR data.
4.9.1.1. From the “Experiments” drop-down menu, select the option “Create
new composite data set…”, enter a name (e.g. VNTR_cmp), and click
the “OK” button. The “Composite data set VNTR_cmp” window will
appear.
4.9.1.2. Highlight the experiment VNTR_vals and from the “Experiment”
drop-down menu, select the option “Use in composite data set”. Close
the window.
4.9.1.3. NOTE: the created composite dataset will be automatically saved in
the database, and hence only need to be created once before the first
analysis in the database
4.9.2. Next time a comparison window is opened (see below “Analysis of the
VNTR data”), there will be a new experiment VNTR_cmp listed in the
bottom of the window (BioNumerics versions lower than 5.0) or in the top
left corner of the window (BioNumerics version 5.0). This experiment will
facilitate the display of a spreadsheet-like view of the copy numbers that
can be shown next to a dendrogram analysis of the data set.
4.9.2.1 NOTE: it may be necessary to scroll the experiment list to the right
with the arrow button to bring VNTR_cmp in display in BioNumerics
versions lower than 5.0).
4.10. Analysis of the VNTR data
4.10.1. The VNTR data contained in the character set VNTR_vals can be analysed
in BioNumerics with all the tools that are available to character data. That
includes cluster analysis with a variety of methods and similarity
coefficients. For VNTR data, the co-efficients that make most sense are:
4.10.1.1. Categorical: preferred if differences in copy numbers should be
treated in a qualitative way.
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4.10.1.2. Manhattan: preferred if differences in copy numbers should be
treated in a quantitative way (larger difference means more distantly
related organisms). This coefficient can be used to construct minimum
spanning trees.
4.10.2. In order to create a dendrogram:
4.10.2.1. Select the isolates to be included in the dendrogram
4.10.2.2. From the “Comparison” drop-down menu, select the option
“Create new comparison” and a “Comparison” window will appear
4.10.2.3. Select “VNTR_cmp” from the bottom of the window in
BioNumerics versions lower than 5.0 or from the top left
“Experiments” window in BioNumerics 5.0.
4.10.2.4. From the “Clustering” drop-down menu, select the option
“Calculate…Cluster analysis (similarity matrix)” and a “Composite
data set comparison” dialog box will appear
4.10.2.5. Select “Categorical” for “Multi-state coefficient” and “UPGMA”
for “Dendrogram type”
4.10.2.6. Click on “OK” button to finish the calculations and the
“Comparison” window with the dendrogram will reappear
4.10.2.7. From the “Layout” drop-down menu, select the option “Show
image”
4.10.2.8. From the “Composite” drop-down menu, select the option ”Show
quantification (values)” and the copy numbers will appear next to the
dendrogram
1.10.1 In order to create a minimum spanning tree:
1.10.1.1 Select the isolates to be included in the spanning tree
1.10.1.2 From the “Comparison” drop-down menu, select the option
“Create new comparison” and a “Comparison” window will appear
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1.10.1.3 Select “VNTR_cmp” from the bottom of the window in
BioNumerics versions lower than 5.0 or from the top left
“Experiments” window in BioNumerics 5.0.
1.10.1.4 From the “Clustering” drop-down menu, select the option
“Calculate…Minimum spanning tree (population modeling)” and a
“Minimum spanning tree” dialog box will appear
1.10.1.5 Make sure the default “Manhattan” is checked for “Coefficient”
and click “OK” and the “Minimum spanning tree” window with the
tree will appear.
1.10.1.6 You can find out the content for each node by clicking on them
individually
5. FLOW CHART:
6. BIBLIOGRAPHY:
7. CONTACTS:
7.1 Eija Hyytia-Trees, D.V.M., Ph.D.
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-3672
EHyytia-Trees@cdc.gov
1.2 Ashley Sabol
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-2947
ASabol@cdc.gov
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ANALYSIS OF MLVA DATA OF SALMONELLA ENTERICA
SEROTYPE ENTERITIDIS IN BIONUMERICS-APPLIED
BIOSYSTEMS GENETIC ANALYZER DATA
Effective Date:
06
08
10
1.3 Patti Lafon
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-2828
PLafon@cdc.gov
8. AMENDMENTS:
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 11 of 12
PULSENET STANDARD OPERATING PROCEDURE FOR
ANALYSIS OF MLVA DATA OF SALMONELLA ENTERICA
SEROTYPE ENTERITIDIS IN BIONUMERICS-APPLIED
BIOSYSTEMS GENETIC ANALYZER DATA
CODE:
PND18
Effective Date:
06
08
10
Appendix PND18-1
S. enterica serotype Enteritidis VNTR allele list and corresponding observed
fragment size ranges in the Applied Biosystems Genetic Analyzer 3130XL
(ABISE look-up table)
Note: This table is posted on the CDC Team as a TXT-file so users may save it locally and use it
with the BioNumerics MLVA scripts. Every time the table is updated, a notification will be posted
on CDC Team together with the latest version of the table.
Count
VNTR_1
1
2 163-166
3 170-172
4 177-178
5 184-186
6 190-193
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
198-199
204-205
211-212
232-234
VERSION:
VNTR_2
VNTR_8
346-349
433-436
300-301
308309.5
310.4316
317.5-324.0
324.9330
335-337
342-344
348-351
356-357
363-364
VNTR_6
VNTR_9
VNTR_3
VNTR_5
160-161
181-184
190-192
200-203
172-174
199-201
211-214
178-180
184-185
189-191
408-411
446-447
479-482
259-260
283-284
566-567
196-197
201-203
207-209
213-215
218-221
225-228
232-234
238-239
244-247
249-253
258-259
264-265
268-271
274-281
287-288
REPLACED BY:
AUTHORIZED BY:
Page 12 of 12
PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN
BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND19
Effective Date:
06
08
10
1. PURPOSE: to describe the standardized protocol for analysis of MLVA data of
Salmonella enterica serotype Enteritidis in BioNumerics.
2. SCOPE: to provide PulseNet participants with a single protocol for analyzing MLVA
data of S. enterica serotype Enteritidis, thus ensuring inter-laboratory comparability
of the generated results.
3. DEFINITIONS:
3.1. MLVA: Multiple-locus variable-number tandem repeat analysis
3.2. VNTR: Variable-number tandem repeat
3.3. CDC: Centers for Disease Control and Prevention
3.4. SOP: Standard Operating Procedure
4. RESPONSIBILITIES/PROCEDURE:
4.1 Software needed for data analysis
4.1.1. BioNumerics version 4.5 or higher
4.1.2. Customized scripts for data import (VNTRImport_v4.bns), copy number
calculation (= allele assignment) (VNTRCalc_v4.bns), troubleshooting
(VNTRReport.bns), and support functions (VNTRDetails_v2.bns)
4.1.3 Look-up table (BeckmanSE.txt) for allele size ranges
4.2 General overview
4.2.1 The import process consists of three major steps:
4.2.1.1 Exporting the appropriate data file from the Beckman system (refer
to SOP PNL27 step 4.10.1.6)
4.2.1.2 Importing this file into BioNumerics (using the script
VNTRImport_v4.bns)
4.2.1.3 Determining the copy numbers (assigning alleles) for each VNTR
(using the script VNTRCalc_v4.bns and the look-up table
BeckmanSE.txt)
4.2.1.3.1 VNTRCalc_v4.bns script facilitates the allele assignment in
two different ways:
4.2.1.3.1.1 “BeckmanSE” – assigns the copy number based on the
look-up table (Appendix PND19-1). This method
should be used in all routine analysis.
4.2.1.3.1.2 “Predicted” – calculates the copy number based on the
mathematical formula: (Observed fragment size –
offset)/repeat size. This method should only be used
when a new allele (not specified in the look-up table) is
identified
VERSION:
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PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN
BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND19
Effective Date:
06
08
10
4.3 Required import format
4.3.1 The data should be exported from the Beckman software as a CSV
(comma-delimited) text file containing the fragment length information.
Multiple columns will be present in the file but only four columns are of
interest: “RN”, “dye”, “est size (nt)” and “pk height (rfu)”. All other
columns will be ignored by the script during import. The field “RN” should
contain information designating the BioNumerics key number (isolate
identifier) loaded in each lane of the sequencer, as well as which VNTR
mastermix or reaction was loaded. The import script will ignore any text that
appears beyond the dot “.” in the RN field.
4.3.1.1 NOTE: no spaces or underscores are allowed between the strain ID
and the reaction ID or between the reaction ID and the dot “.” in the
RN field.
4.4 Setting up a new database
4.4.1 Click “New” to set up a new database
4.4.2 Type in the name (for example “SEMLVA”) of your new database and
click ”Next”
4.4.3 Select the default directory to set up a new database and click “Next”
4.4.4 Choose “Yes” to enable creation of log files and click “Finish”
4.4.5 Choose the default “Use the local database” and click “OK”
4.4.6 Go to: Program files → BioNumerics → Data → SEMLVA and create
two folders named “Scripts” and “VNTRtables”.
4.4.7 Save the four MLVA specific scripts (VNTRImport_v4.bns,
VNTRCalc_v4.bns, VNTRReport.bns, VNTRDetails_v2.bns) in the
“Scripts” folder and the look-up table (BeckmanSE.txt) in the
“VNTRtables” folder
4.5 Importing a peak file for the first time
4.5.1 Review the peak file: make sure that the observed sizes for the D1 labelled
molecular size standard peaks are within ± 1 bp from the expected size.
Remove any data (failed reactions, controls, internal ladder) that you don’t
want to import in the BioNumerics from the CSV file. Re-name and re-save
the CSV file either on your hard drive or on the flash drive.
4.5.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “Beckman peak file”. The
script pops up a file dialog box, prompting for the name of the file to import.
Select the appropriate file and click “OK”. A second dialog box pops up,
prompting for a variety of other information:
4.5.2.1 Fingerprint file name. This is the name of the fingerprint file that
will be used in BioNumerics. Leave this unchanged, unless a file
has already been imported with the same name. In this case, change
the file name into a new, unique and informative name.
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Page 2 of 10
PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN
BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND19
Effective Date:
06
08
10
4.5.2.2 Dyes to import. The first dye (D1) contains only reference markers
and does not need to be imported. Use this list to select what dyes
will be imported; they are D2, D3 and D4. Each dye will be stored
in a different file, resulting from appending the dye name to the
fingerprint file name.
4.5.2.3 Assign reference positions. Leave this option checked.
4.5.2.4 Select imported isolates. Leave this option checked.
4.5.2.5 Pool tags. For this protocol, there are two pool tags representing the
two PCR reactions for each isolate (all loaded on different wells)
and tagged with names “R1” and “R2”. The two tags should appear
in the list. If they do not, they can be added by selecting “Add” to
update.
4.5.2.6 If everything is filled appropriately, click “OK” to start the import.
4.5.2.7 NOTE: These settings are automatically saved and reloaded the
next time this script is run.
4.5.3
When the script is finished, it has:
4.5.3.1 Created a fingerprint type “VNTRFpr” with appropriate settings
4.5.3.2 Created fingerprint types with appropriate settings for each dye and
each pool used. The names of these fingerprint types are
“VNTRFpr”+“pool name”+“dye” (for example: VNTRFprR1_D2).
4.5.3.3 Created fingerprint files for each dye in the fingerprint type
“VNTRFpr,” and imported the band information in these lanes. The
individual lanes are assigned to their appropriate pool fingerprint
type, according to the known pool tags found in the “RN” field.
4.5.3.4 Created new database entries for all isolates found
4.5.3.5 Linked the fingerprints to their corresponding entries
4.5.3.6 Selected all isolates for which fingerprint data was imported
4.6 Determining the copy numbers for the first time (allele assignment)
4.6.1 Run the script VNTRCalc_v4 from the scripts menu. The first time this
script is run, it will automatically create a new character set called
“VNTR_vals”. This character set will hold the copy numbers for each
VNTR. The script will also create a second character set called
“VNTR_frags” which holds the fragment size with a highest fluorescence for
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 3 of 10
PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN
BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND19
Effective Date:
06
08
10
each VNTR. Click “OK” on the two pop-up windows notifying that these
two character types will be created.
4.6.2 Before the script can determine copy numbers from the fragment length
information, it should have sufficient information about what VNTRs can be
found in which pool and dye, what are the repeat lengths, etc. To this end,
the script pops up a dialog box called “VNTR assign”. This dialog box
contains a list of defined VNTRs that is initially empty. To enter the
parameters of the first VNTR, click on “Add”. This brings up a second
dialog box, “Edit VNTR”, prompting for all properties of this first VNTR.
Refer to the laboratory protocol PNL27, appendix PNL27-4 for all necessary
information to load the specifications for each VNTR.
4.6.3
The VNTR information includes:
4.6.3.1 Name. This name will be used for further reference and for the
character name in the character type VNTR_vals. Each VNTR
should have a unique name (for example VNTR_1, VNTR_2, etc).
4.6.3.2 Offset. Each fragment consists of a repeat portion and a constant
portion, due to the fact that the primers do not locate exactly at the
start and the end of the repeat region. This parameter specifies the
size (in base pairs) of the constant portion.
4.6.3.3 Repeat length. This parameter specifies the size of the repeat block
unit (in base pairs).
4.6.3.4 Copy range. These parameters specify the minimum and maximum
number of copies that the script will consider during the copy
number determination.
4.6.3.5 Tolerance. This specifies the maximum difference between the
expected fragment length (calculated by the software) and the actual
length estimated by the sequencer.
4.6.3.6 Take from fingerprint type. This drop-down list should be used to
indicate on what fingerprint type this VNTR was run. The
fingerprint type is determined by the dye and the pool tag.
4.6.4 If all parameters are filled in appropriately, click OK to add the VNTR and
return to the previous dialog box (“VNTR assign”). Repeat the same actions
to enter the information for all VNTRs used for this protocol. When all
VNTRs are entered, make sure the box for “Code absent as negative” is
VERSION:
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Page 4 of 10
PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN
BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND19
Effective Date:
06
08
10
checked. Select “BeckmanSE” from the “Fragment ranges” dropdown menu
and press the “Save&Assign” button to let the script assign copy numbers
for the currently selected entries.
4.6.4.1 NOTE 1: pressing “Save&Quit” would store the information
without assigning copy numbers for the current selection.
4.6.4.2 NOTE 2: all information regarding the VNTRs is stored with the
database. The next time the script is run, the VNTR definitions will
be loaded automatically. New incoming VNTR data can be
processed simply by selecting the corresponding data entries,
launching the script VNTRCalc_v4 from the “Scripts” drop-down
menu, and pressing the button “Save&Assign”.
4.6.4.3 For every fragment with the highest fluorescence level in the
fingerprint type (= the combination of the reaction and the dye), the
script will assign an allele type (=a copy number) based on the
fragment size ranges specified in the “BeckmanSE” look-up table
(Appendix PND19-1). Note that, within the same fingerprint type,
more than one VNTR can be loaded if there is no overlap within the
fragment size ranges.
4.6.5 When the script has completed, copy numbers for all VNTRs for the
currently selected entries are determined.
4.6.5.1 Note: Two types of problems may arise during the process (if one or
more such errors were encountered during the assignment, an error
report is displayed listing all the problems):
4.6.5.1.1 None of the peaks present in a fingerprint are compatible
with the fragment size ranges in the look-up table. In this
case, the corresponding character value will be scored “2.0”. In this situation, run the “VNTRReport” script from the
“Scripts” drop-down menu to verify whether the problem
occurred because the VNTR failed to amplify (mutations,
insertions or deletions in the primer annealing region, the
whole VNTR array and the flanking sequences missing,
VNTR located on a plasmid) or because the VNTR had a
fragment size outside the acceptable range (incomplete
repeats, new alleles). In the latter case, rerun the reaction to
confirm the accuracy and reproducibility of the sizing. The
isolate should be submitted to CDC for confirmation if the
size upon retesting is still outside the fragment range
specified in the look-up table.
4.6.5.1.2 More than one peak in a fingerprint is compatible with an
acceptable fragment size range. In this case, the script will
use the solution that corresponds to the peak with the largest
“peak height” value.
4.7 Importing a peak file and assigning alleles on a routine basis
VERSION:
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Page 5 of 10
PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN
BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND19
Effective Date:
06
08
10
4.7.1
Review the peak file: make sure that the observed sizes for the D1 labelled
molecular size standard peaks are within ± 1 bp from the expected size.
Remove any data (failed reactions, controls, internal ladder) that you don’t
want to import in the BioNumerics from the CSV file. Re-name and re-save
the CSV file either on your hard drive or on the flash drive.
4.7.2 In BioNumerics, run the script VNTRImport_v4 from the “Scripts” dropdown menu. The “Import VNTR peak data” dialog box will appear. From
the “Peak file format” drop-down menu, select “Beckman peak file”. The
script pops up a file dialog box, prompting for the name of the file to import.
Select the appropriate file and click “OK”. “VNTR/Import peak data” dialog
box will appear. De-select “D1” from the “Dyes to import” and click “OK”.
4.7.3 Run the script VNTRCalc_v4 from the scripts menu. “VNTR assign”
dialog box will appear. Select “BeckmanSE” from the “Fragment ranges”
drop-down menu and click “Save&Assign” to assign allele numbers.
4.8 Verifying the allele assignment
4.8.1 For each isolate in the database that has some VNTR data associated, you
can click on the “VNTR frags” and “VNTR vals” entries in the list to open
the “Entry edit” windows.
4.8.2
If the VNTRCalc_v4 script did not detect a fragment for a VNTR, refer to
the step 4.6.5.1.1 of this protocol for details on how to proceed. If the
fragment size is slightly outside the range specified in the “BeckmanSE”
look-up table it is possible to manually assign a temporary allele size and
type for that VNTR until an official confirmation has been performed by
CDC (Note: only the CDC database managers are allowed to modify the
BeckmanSE.txt file. Once a modification has been made, the modified file
will be posted on CDCTeamSupport along with a notification that the file
has been modified). To manually assign an allele size and type click on the
fragment size in the “VNTRfrags” entry edit window or the allele type in the
“VNTR vals” entry edit window. This will open the “Change character
value” window in BioNumerics lower than 5.0. In BioNumerics 5.0, the
allele size or type can be directly highlighted and changed in the entry edit
window (Screen shots below)
VERSION:
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Page 6 of 10
PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN
BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND19
Effective Date:
06
08
10
4.9 Visualizing the data
4.9.1 In order to display copy numbers next to a dendrogram in a comparison,
first create a “composite data set” that holds the VNTR data.
4.9.1.1 From the “Experiments” drop-down menu, select the option “Create
new composite data set…”, enter a name (e.g. VNTR_cmp), and
click the “OK” button. The “Composite data set VNTR_cmp”
window will appear.
4.9.1.2 Highlight the experiment VNTR_vals and from the “Experiment”
drop-down menu, select the option “Use in composite data set”.
Close the window.
4.9.2 Next time a comparison window is opened (see below “Analysis of the
VNTR data”), there will be a new experiment VNTR_cmp listed in the
bottom of the window (BioNumerics versions lower than 5.0) or in the top
left corner of the window (BioNumerics version 5.0). This experiment will
facilitate the display of a spreadsheet-like view of the copy numbers that can
be shown next to a dendrogram analysis of the data set.
4.9.2.1 NOTE: it may be necessary to scroll the experiment list to the right
with the arrow button to bring VNTR_cmp in display in BioNumerics
versions lower than 5.0.
4.10 Analysis of the VNTR data
4.10.1. The VNTR data contained in the character set VNTR_vals can be analysed
in BioNumerics with all the tools that are available to character data. That
includes cluster analysis with a variety of methods and similarity
coefficients. For VNTR data, the coefficients that make most sense are:
4.10.1.1 Categorical: preferred if differences in copy numbers should be
treated in a qualitative way. This is the only option for creating
dendrograms using MLVA data.
4.10.1.2 Manhattan: preferred if differences in copy numbers should be
treated in a quantitative way (larger difference means more distantly
related organisms). This coefficient can be used to construct
minimum spanning trees.
4.10.2 In order to create a dendrogram:
4.10.2.1 Select the isolates to be included in the dendrogram
VERSION:
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Page 7 of 10
PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN
BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND19
Effective Date:
06
08
10
4.10.2.2 From the “Comparison” drop-down menu, select the option
“Create new comparison” and a “Comparison” window will appear
4.10.2.3 Select “VNTR_cmp” from the bottom of the window in
BioNumerics versions lower than 5.0 or from the top left
“Experiments” window in BioNumerics 5.0.
4.10.2.4 From the “Clustering” drop-down menu, select the option
“Calculate…Cluster analysis (similarity matrix)” and a “Composite
data set comparison” dialog box will appear
4.10.2.5 Select “Categorical” for “Multi-state coefficient” and “UPGMA”
for “Dendrogram type”
4.10.2.6 Click on “OK” button to finish the calculations and the
“Comparison” window with the dendrogram will reappear
4.10.2.7 From the “Layout” drop-down menu, select the option “Show
image”
4.10.2.8 From the “Composite” drop-down menu, select the option ”Show
quantification (values)” and the copy numbers will appear next to
the dendrogram
4.10.3 In order to create a minimum spanning tree:
4.10.3.1 Select the isolates to be included in the spanning tree
4.10.3.2 From the “Comparison” drop-down menu, select the option
“Create new comparison” and a “Comparison” window will appear
4.10.3.3 Select “VNTR_cmp” from the bottom of the window in
BioNumerics versions lower than 5.0 or from the top left
“Experiments” window in BioNumerics 5.0.
4.10.3.4 From the “Clustering” drop-down menu, select the option
“Calculate…Minimum spanning tree (population modeling)” and a
“Minimum spanning tree” dialog box will appear
4.10.3.5 Make sure the default “Manhattan” is checked for “Coefficient”
and click “OK” and the “Minimum spanning tree” window with the
tree will appear.
VERSION:
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Page 8 of 10
PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN
BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND19
Effective Date:
06
08
10
4.10.3.6 You can find out the content for each node by clicking on them
individually.
5. FLOW CHART:
6. BIBLIOGRAPHY:
7. CONTACTS:
7.1. Eija Hyytia-Trees, D.V.M., Ph.D.
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-3672
EHyytia-Trees@cdc.gov
7.2. Ashley Sabol
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-2947
ASabol@cdc.gov
7.3.. Patti Lafon
PulseNet Methods Development and Reference Unit, EDLB, CDC
(404) 639-2828
PLafon@cdc.gov
8. AMENDMENTS:
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 9 of 10
PULSENET STANDARD OPERATING PROCEDURE FOR ANALYSIS OF
MLVA DATA OF SALMONELLA ENTERICA SEROTYPE ENTERITIDIS IN
BIONUMERICS-BECKMAN COULTER CEQ 8000 DATA
CODE: PND19
Effective Date:
06
08
10
Appendix PND19-1
S. enterica serotype Enteritidis VNTR Allele List and Corresponding
Observed Fragment Sizes in the Beckman Coulter CEQ 8000
(BeckmanSE look-up table)
Note: This is posted on the CDCTeam as a TXT-file so users may save it locally and use it with
the BioNumerics MLVA scripts. Every time the table is updated, a notification will be posted on
CDC Team together with the latest version of the table.
Count
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
VNTR_1
166-168
172-174
180-181
185-189
193-196
200-202
207-209
214-215
236-237
VERSION:
VNTR_2
295-296
299-302
305-309
312-315
318-322
325-329
333-335
338-341
346-349
353-354
VNTR_8
344-347
433-435
VNTR_6
410-413
443-445
476-481
VNTR_9
VNTR_3
VNTR_5
162-164
183-186
192-195
199-200
197-201
209-213
171-172
177-179
183-185
189-191
195-197
201-203
207-209
213-215
219-221
225-227
231-233
237-239
243-245
249-250
255-257
260-263
266-267
272-274
258-259
280-282
569-570
284-285
REPLACED BY:
AUTHORIZED BY:
Page 10 of 10
READING CONTROL SHEET FOR:
STANDARD OPERATING PROCEDURES FOR THE PULSENET DATABASES
(PND)
NAME
DATE
COMMENTS
SIGNATURE
By signing above, you are indicating that you have read and understood all SOPs
included in the PND section of this manual.
CODE: PNQ01
STANDARD OPERATING PROCEDURE FOR TIFF QUALITY GRADING
Effective Date:
5
09
2005
1.
PURPOSE: To describe guidelines for the quality of TIFF images submitted to the PulseNet national
databases.
2.
SCOPE: This applies to all TIFF images submitted to PulseNet, thereby allowing comparison of results
with other PulseNet laboratories.
3.
DEFINITIONS/TERMS:
3.1 TIFF: Tagged Image File Format
3.2 TIFF Quality: The grading of the appearance and ease of analysis of a TIFF, according to the TIFF
Quality Grading Guidelines within this SOP. This is a main component of the evaluation of a TIFF
submitted for certification or proficiency testing.
3.3 SOP: Standard Operating Procedure
4.
RESPONSIBILITIES/PROCEDURE:
TIFF Quality Grading Guidelines
Parameter
Excellent
Good
Fair
Poor
By protocol, for
example:
- Gel fills whole
TIFF
- Wells included on
TIFF
- Bottom band of
standard 1-1.5 cm
from bottom of gel
The cell
concentration is
approximately the
same in each lane
- Gel doesn’t fill
whole TIFF but band
finding is not affected
Not protocol; for example, >1
of the following:
- Gel doesn’t fill whole TIFF
and this affects band finding
- Wells not included on TIFF
- Bottom band of standard not
1-1.5 cm from bottom of gel
- Band spacing of standards
doesn’t match global standard
The cell concentrations are
uneven from lane to lane,
making the gel impossible to
analyze
Bands
Clear and distinct
all the way to the
bottom of the gel
Lanes
Straight
- Slight band
distortion in 1 lane
but doesn’t interfere
with analysis
- Bands are slightly
fuzzy and/or slanted
- A few bands (e.g.,
≤3) difficult to see
clearly (e.g., DNA
overload), especially
at bottom of gel
- Slight smiling
(higher bands in the
outside lanes vs. the
inside)
- Lanes gradually run
longer toward the
right or left
- Still analyzable
Not protocol; for example,
one of the following:
- Gel doesn’t fill whole TIFF
and band finding is affected
- Wells not included on TIFF
- Bottom band of standard not
1-1.5 cm from bottom of gel
- Band spacing of standards
doesn’t match global standard
- >2 lanes contain darker or
lighter bands than the other
lanes, or
- At least 1 lane is much
darker or lighter than the
other lanes, making the gel
difficult to analyze
- Some band distortion (e.g.,
nicks) in 2-3 lanes but still
analyzable
- Fuzzy bands
- Some bands (e.g., 4-5) are
too thick
- Bands at the bottom of the
gel are light, but analyzable
- Significant smiling
- Slight curves on the outside
lanes
- Still analyzable
- Smiling or curving that
interferes with analysis
Image
Acquisition
and Running
Conditions
Cell
Suspensions
VERSION:
1-2 lanes contain
darker or lighter
bands than the other
lanes
REPLACED BY:
- Band distortion that makes
analysis difficult
- Very fuzzy bands.
- Many bands too thick to
distinguish
- Bands at the bottom of the
gel too light to distinguish
AUTHORIZED BY:
Page 1 of 2
CODE: PNQ01
STANDARD OPERATING PROCEDURE FOR TIFF QUALITY GRADING
Effective Date:
5
09
2005
Restriction
Complete
restriction in all
lanes
- One to two faint
shadow bands on gel
- One lane with many shadow
bands
- A few shadow bands spread
out over several lanes
- Greater than 1 lane with
several shadow bands
- Lots of shadow bands over
the whole gel
Gel
Background
Clear
- Mostly clear
background
- Minor debris
present that doesn’t
affect analysis
- Some debris present that
may or may not make
analysis difficult (e.g., auto
band search finds too many
bands)
- Background caused by
photographing a gel with very
light bands (image contrast
was “brought up” in
photographing gel-makes
image look grainy)
- Lots of debris present that
may or may not make
analysis difficult (i.e., auto
band search finds too many
bands)
DNA
Degradation
Not present
- Minor background
(smearing) in a few
lanes but bands are
clear
- Significant smearing in 1-2
lanes that may or may not
make analysis difficult
- Minor background
(smearing) in many lanes
- Significant smearing in >2
lanes that may or may not
make analysis difficult
- Smearing so that a lane is
not analyzable (except if
untypeable [thiourea
required])
(smearing in
the lanes)
5.
FLOW CHART:
6. BIBLIOGRAPHY:
7. CONTACTS:
8. AMENDMENTS:
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 2 of 2
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
CODE: PNQ02
Effective Date:
04
26
10
1. PURPOSE: To describe the procedure for certifying PulseNet personnel to enable full
participation in PulseNet activities, including on-line access to the PulseNet National
Databases.
2. SCOPE: This procedure applies to all PulseNet personnel performing PFGE, image
acquisition, and/or BioNumerics gel analysis.
3. DEFINITIONS/TERMS:
3.1.
3.2.
3.3.
3.4.
3.5.
PFGE: Pulsed-field Gel Electrophoresis
CDC: Centers for Disease Control and Prevention
QA/QC: Quality Assurance/Quality Control
SOP: Standard Operating Procedure
BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths,
Belgium
3.6. TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in
BioNumerics
3.7. Bundle file: A file with a .bdl extension that is produced in BioNumerics and contains the
analysis of at least one lane of a gel image
3.8. Gel-certified or TIFF-certified: An individual or laboratory that is certified in laboratory
methods for PFGE and image acquisition
3.9. Analysis-certified: An individual who is certified in BioNumerics gel analysis
3.10. Certification files: TIFF and/or bundle files submitted by PulseNet participants for
certification evaluation.
3.11. Certification file evaluator: An individual who evaluates certification files
3.12.PulseNet Area Laboratory: Laboratory, designated by CDC, which has agreed to assume
responsibility for additional PulseNet duties for laboratories within their support zone. The
current Area Laboratories include MA, MN, WA, TX, VA, UT, MI, and CDC.
4. RESPONSIBILITIES:
4.1 Individuals performing PulseNet-related work (i.e., preparing PFGE gels and/or analyzing TIFFs of
PFGE gels) must submit certification file(s) and have them reviewed before being able to submit
TIFF images and BioNumerics analyses to the PulseNet National Databases.
4.1.1 Submitted certification files must document the submitter’s highest level of competence in
producing and imaging PFGE gels and/or in analyzing TIFFs of PFGE gels.
4.1.2 Individuals can be certified for each PulseNet organism in one of three ways:
4.1.2.1 Gel only (i.e., gel-certified). An individual can submit TIFFs to an analysis-certified person
for subsequent analysis and uploading to the national databases.
4.1.2.2 Analysis only (i.e., analysis-certified). An individual can analyze TIFFs of gels generated
by TIFF-certified individuals and upload those analyzed images to the national databases.
4.1.2.3 Both gel and analysis (i.e., gel and analysis-certified). An individual can perform PFGE,
image acquisition, and BioNumerics analysis of TIFFs, uploading the analyses to the
national databases.
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 1 of 27
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
CODE: PNQ02
Effective Date:
04
26
10
4.1.3 For each PulseNet organism, at least one person from each PulseNet participating laboratory
should be gel-certified and one person should be analysis-certified. One person can be both gel
and analysis-certified. Laboratories cannot have analysis-certified personnel without gel-certified
personnel; gel certification must occur before or concurrent with analysis certification.
5.
PROCEDURE:
5.1 PulseNet participants request certification set(s) from CDC (via pfge@cdc.gov) if they do not already
have them.
5.1.1 Currently, certification sets are available for E. coli O157:H7, E. coli Non O157 STEC (analysisonly), Salmonella, Shigella, Listeria monocytogenes, Campylobacter jejuni, Vibrio cholerae,
Vibrio parahaemolyticus and C.botulinum.
5.2 CDC sends the requested certification set(s) and detailed instructions for the gel preparation and
analysis of the certification set organisms. See Appendices PNQ02-1 through PNQ02-9.
5.3 Individuals in each laboratory who wish to be gel-certified make plugs for each sample and run gel(s)
with the proper controls.
5.4 Individuals in the laboratory who wish to be analysis-certified analyze TIFF(s) produced using the
certification set instructions and create a bundle file of their analyses, according to the certification set
instructions.
5.5 TIFF file(s) are submitted to CDC for review. See Appendices PNQ02-1 through PNQ02-9 for
submission instructions.
5.6 Bundle files are submitted to CDC for review. See Appendices PNQ02-1 through PNQ02-9 for
submission instructions.
5.7 Submitters are notified in writing of the results of their certification file(s) evaluation. (See PNQ03
for more information on the evaluation of PulseNet certification files.)
5.7.1 If the submitted certification files pass the certification evaluation, the submitter is considered
certified as long as they remain in their current laboratory and that laboratory successfully
completes annual proficiency testing (See PNQ04 for more information on the PulseNet
proficiency testing program). If a person relocates to a different PulseNet laboratory, they must be
recertified.
5.7.2 If the submitted certification files do not pass the certification evaluation
5.7.2.1 The individual will need to review the troubleshooting comments received from the
evaluator and resubmit once results have improved
5.7.2.2 At any point, if the evaluator and/or CDC feels that the individual needs additional
troubleshooting and/or training before resubmission, the Area Laboratory responsible for
the individual’s region will be notified
5.7.2.3 If the submitter fails certification three times, the individual will not be allowed to submit
again for six months. Before resubmitting, the individual will be expected to work with
CDC and/or their PulseNet Area Laboratory until satisfactory results are achieved. This
includes, but is not limited to troubleshooting and training in the PulseNet PFGE protocols,
BioNumerics and the PulseNet masterscripts.
6. FLOW CHART:
7. BIBLIOGRAPHY:
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 2 of 27
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
CODE: PNQ02
Effective Date:
04
26
10
8. CONTACTS:
8.1 PulseNet Database Unit Chief: Kelley Hise, MPH
(404) 639-0704
KHise@cdc.gov
8.2 CDC PulseNet Database Administration Team
(404) 639-4558
PFGE@cdc.gov
9. AMENDMENTS:
9.1 Appendix PNQ02-6 added on 5/2/2007
9.2 Appendix PNQ02-7 added on 11/18/2008
9.3 Appendix PNQ02-8 added on 10/6/2009
9.4 As of 10/6/2009 the Shigella certification set (Appendix PNQ02-3) no longer includes isolates 92-01,
93-01 or 96-01.
9.5 As of 10/6/2009 the Campylobacter jejuni certification set (Appendix PNQ02-5) no longer includes
isolates D996 or D2253.
9.6 1/5/2010 Appendix PNQ02-6 section 4 was updated to correct lane information for the test strains
9.7 Appendix PNQ02-9 added on 4/22/2010
Appendix PNQ02-1
Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) E. coli O157:H7 Cultures
Biological Safety Warning: E. coli O157:H7 strains are considered Level 2 biological agents by the U.S.
Department of Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry out
laboratory work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.
Note: Store the lyophilized cultures at 4oC in the dark until they are reconstituted.
Materials Needed:
Sturdy sterile forceps
1 ml pipetman
1 ml sterile pipet tips
1 ul sterile inoculating loop
Reagents Needed:
Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
Sterile grade reagent water
70% isopropyl alcohol
Procedure for Reviving Cultures:
Day 1
1. Document the isolate number(s) and the date(s) lyophilized for your records. Wipe the aluminum cover and
outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber
stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial
with isopropyl alcohol before removing the stopper.
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 3 of 27
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
CODE: PNQ02
Effective Date:
04
26
10
2. Re-suspend the lyophilized cells with 1ml of sterile grade reagent water. Allow to stand for a few minutes and/or
mix gently to produce a uniform suspension. With an inoculating loop, streak a small amount of this suspension onto
a blood agar plate (BAP) and incubate at 37ºC overnight.
Days 2 and 3
Check the BAP; if the culture appears pure, pick an isolated colony, and inoculate a fresh BAP for heavy growth;
incubate at 37oC overnight. Use the growth from this plate to make PFGE plugs of the standard strains. Before
making the plugs, transfer culture to fresh medium and incubate at 37oC overnight; this will ensure that the same
culture can be retested, if necessary.
After the strains have been reconstituted according to the above directions, streak each culture to agar plates, pick an
isolated colony, and subculture to another plate. Use the growth from the second plate to make the PFGE plugs.
Please let me know if the package does not arrive in satisfactory condition, or if the cultures are not viable. Freeze
(-70°C) or stock these strains according to your laboratory’s policy within one week of receiving them. Then
your laboratory will have stock cultures of this PulseNet certification set for future use, including the
PulseNet certification of additional personnel.
The strain numbers of the E. coli cultures are as follows:
CDC16-98
CDC20-98
CDC24-98
CDC68-98
Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Escherichia coli,
non-typhoidal Salmonella serotypes and Shigella sonnei by Pulsed Field Gel Electrophoresis (PFGE)”. Detailed
instructions for making the PFGE plugs can be found in the PulseNet QA/QC Manual PNL05. This document is
available in the QA/QC conference on CDC Team or at www.cdc.gov/pulsenet/. If you can not access the protocol,
please request it by sending an E-mail to PFGE@CDC.GOV.
1. Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they
can be retested several times, if necessary.
2. Restrict one plug slice from the four test cultures (CDC16-98, CDC20-98, CDC24-98, and
3.
4.
CDC68-98) and 3 plug slices of Salmonella ser. Braenderup H9812 (the PulseNet Universal
Standard Strain) with XbaI (40-50 Units/plug slice) for 2 hours at 37°C..
Restrict one plug slice from three of the test cultures (CDC16-98, CDC20-98, and CDC24-98)
with AvrII (BlnI; 25-30 Units/plug slice) for 2 hours at 37°C.
Load H9812 in Lanes 1, 5, 10 and the test strains as follows:
Lane
Certification Strain
Enzyme
1
H9812
XbaI
2
16-98
XbaI
3
20-98
XbaI
4
24-98
XbaI
5
H9182
XbaI
6
16-98
BlnI
7
20-98
BlnI
8
24-98
BlnI
9
68-98
XbaI
10
H9182
XbaI
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 4 of 27
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
CODE: PNQ02
Effective Date:
04
26
10
Note: If you use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty.
Although the use of the ~0.5-mm wide 15-teeth combs [Bio-Rad, 170-4326] in the standard casting stand
[14 x 13-cm] is not recommended for routine PFGE analysis of test isolates, the use of the smaller comb
teeth will be allowed for certification.
5. Run the gel using the E. coli electrophoresis conditions. These run times are based on the
equipment and reagents used at CDC. If the gels generated in your lab do not have the lowest
band in strain H9812 approximately 1-1.5 cm from the bottom of the gel, the run time may have to
be changed.
6. Staining, de-staining, and gel documentation (imaging):
a. The gel image should fill the entire window of the imaging equipment (computer) screen
(without cutting off wells or lower bands). Individuals submitting certification TIFFs that do
not contain the wells or clearly show the bottom bands of the patterns that will automatically
be asked to rerun the gel and submit a new certification TIFF.
b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more
shadow bands (indicating incomplete restriction) should not be submitted for certification. To
eliminate incomplete restriction wash the plugs at least two more times with TE Buffer before
restriction is repeated. If the problem persist repeat the restriction with more units of enzyme,
for a longer amount of time and/or with a different lot of enzyme..
7. Bundle file creation
a.
In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC
strain number in the BioNumerics “Key” field. The bundle file should contain 4 database
entries as follows: 3 test strains restricted with XbaI and BlnI and one test strain restricted
only with XbaI. If there are more than 4 entries in the bundle file, one or more lanes have not
been linked properly.
b.
Create the bundle file using the lightning bolt icon.(refer to the Gel Analysis Guidelines
PND04 for instructions on how to create a PulseNet Bundle File). Bundles created using this
icon are “PulseNet bundles” and will contain only standardized PulseNet experiments and
fields. “PulseNet bundles” can be recognized by the “PN” that is automatically added to the
file name. If a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.
8. Send the TIFF file and/or bundle (*.bdl) file of your gel images to CDC Pulse Net at PFGE@CDC.GOV
within four weeks after receiving the strains.
a. In the email to CDC, include E. coli Certification Gel in the Subject line, so it can be forwarded to the
correct person. Include the isolate number and restriction enzyme used for each lane on the gel in the body
of the email. If you are sending certification TIFFs and bundle files for more than one organism, please
send one email per organism.
b. Name the TIFF and bundle files of your gel images as follows:
TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two to
four letters of the file. The next 2 spaces will indicate the year the file was created. The
next 3 spaces indicate the sequential number of the file submitted from your laboratory
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 5 of 27
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
CODE: PNQ02
Effective Date:
04
26
10
during a calendar year. For example: GA09012.tif is the twelfth file submitted from the
state of Georgia during 2009.
Bundle: The bundle files are named in a similar manner as the TIFF files with the first two
to four letters of the file name indicating the unique identifier code for your laboratory. The
next 2 spaces indicate the year the bundle file was created, and the next 3 spaces indicate
the sequential bundle file number from each laboratory. For example, the eighth bundle file
submitted from the state of Georgia during 2009 would be named GA09008PN.bdl.
For each PulseNet pathogen, an individual may be certified for:
a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.
After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts.
Appendix PNQ02-2
Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Salmonella Cultures
Biological Safety Warning: Salmonella strains are considered Level 2 biological agents by the U.S. Department of
Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry out laboratory
work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.
Note: Store the lyophilized cultures at 4oC in the dark until they are reconstituted.
Materials Needed:
Sturdy sterile forceps
1 ml pipetman
1 ml sterile pipet tips
1 ul sterile inoculating loop
Reagents Needed:
Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
Sterile grade reagent water
70% isopropyl alcohol
Procedure for Reviving Cultures:
Day 1
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 6 of 27
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
CODE: PNQ02
Effective Date:
04
26
10
1. Document the isolate number(s) and the date(s) lyophilized for your records. Wipe the aluminum cover and
outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber
stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial
with isopropyl alcohol before removing the stopper.
2. Re-suspend the lyophilized cells with 1ml of sterile grade reagent water. Allow to stand for a few minutes and/or
mix gently to produce a uniform suspension. With an inoculating loop, streak a small amount of this suspension onto
a blood agar plate (BAP) and incubate at 37ºC overnight.
Days 2 and 3
1. Check the BAP; if the culture appears pure, pick an isolated colony, and inoculate a fresh BAP for heavy growth;
incubate at 37oC overnight. Use the growth from this plate to make PFGE plugs of the standard strains. Before
making the plugs, transfer culture to fresh medium and incubate at 37oC overnight; this will ensure that the same
culture can be retested, if necessary.
After the strains have been reconstituted according to the above directions, streak each culture to agar plates, pick an
isolated colony, and subculture to another plate. Use the growth from the second plate to make the PFGE plugs.
Please let me know if the package does not arrive in satisfactory condition, or if the cultures are not viable. Freeze
(-70°C) or stock these strains according to your laboratory’s policy within 1 week of receiving them. Then,
your laboratory will have stock cultures of this PulseNet certification set for future use, including the
PulseNet certification of additional personnel.
The strain numbers of the Salmonella cultures are as follows:
CDC61-99
CDC78-99
CDC87-03
CDC98-03
Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Escherichia coli,
non-typhoidal Salmonella serotypes and Shigella sonnei by Pulsed Field Gel Electrophoresis (PFGE).” Detailed
instructions for making the PFGE plugs can be found in the PulseNet QA/QC Manual PNL05. This document is
available in the QA/QC conference on CDC Team or at www.cdc.gov/pulsenet/. If you can not access the protocol,
please request it by sending an E-mail to PFGE@CDC.GOV.
1. Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they
can be retested several times, if necessary.
2. Restrict one plug slice from the four test cultures (CDC61-99, CDC78-99, CDC87-03, and
3.
4.
CDC98-03) and 3 plug slices of Salmonella ser. Braenderup H9812 (the PulseNet Universal
Standard Strain) with XbaI (40-50 Units/plug slice) for 2 hours at 37°C.
Restrict one plug slice from three of the test cultures (CDC61-99, CDC78-99, and CDC87-03)
with AvrII (BlnI; 25-30 Units/plug slice) for 2 hours at 37°C.
Load H9812 in Lanes 1, 5, 10 and the test strains as follows:
VERSION:
Lane
Certification Strain
Enzyme
1
H9812
XbaI
2
61-99
XbaI
3
78-99
XbaI
4
87-03
XbaI
5
H9182
XbaI
6
61-99
BlnI
7
78-99
BlnI
REPLACED BY:
AUTHORIZED BY:
Page 7 of 27
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
8
9
10
87-03
98-03
H9182
CODE: PNQ02
Effective Date:
04
26
10
BlnI
XbaI
XbaI
Note: If you use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty.
(The use of the ~0.5-mm wide 15-teeth combs [Bio-Rad, 170-4326] in the standard casting stand [14 x 13cm] is not recommended for routine PFGE analysis of test isolates, but will be allowed for certification).
5. Run the gel using the Salmonella electrophoresis conditions. The run times listed in the protocol
are based on the equipment and reagents used at CDC. If the gels generated in your lab do not
have the lowest band in strain H9812 approximately 1-1.5 cm from the bottom of the gel, the run
time may have to be changed.
6.
Staining, de-staining, and gel documentation (imaging):
a. The gel image should fill the entire window of the imaging equipment (computer) screen
(without cutting off wells or lower bands). Individuals submitting a certification TIFFs that do
not contain the wells or clearly show the bottom bands of the patterns will automatically be
asked to rerun the gel and submit a new certification TIFF.
b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more
shadow bands (indicating incomplete restriction) should not be submitted for certification. To
eliminate incomplete restriction wash the plugs at least two more times with TE Buffer before
restriction is repeated. If the problem persists repeat the restriction with more units of enzyme,
for a longer amount of time and/or with a different lot of enzyme.
7. Bundle file creation
a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC
strain number in the BioNumerics “Key” field. The bundle file should contain 4 database
entries as follows: 3 test strains restricted with XbaI and BlnI and one test strains restricted
only with XbaI. If there are more than 4 entries in the bundle file, one or more lanes have not
been linked properly.
b.
Create the bundle file using the lightning bolt icon.(refer to the Gel Analysis Guidelines
PND04 for instructions on how to create a PulseNet Bundle File). Bundles created using this
icon are “PulseNet bundles” and will contain only standardized PulseNet experiments and
fields. “PulseNet bundles” can be recognized by the “PN” that is automatically added to the
file name. If a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.
8. Send the TIFF file and/or bundle (*.bdl) file of your gel images to CDC Pulse Net at
PFGE@CDC.GOV within four weeks after receiving the strains.
a.
In the email to CDC, include Salmonella Certification Gel in the Subject line, so it can be
forwarded to the correct person. Include the isolate number and restriction enzyme used for
each lane on the gel in the body of the email. If you are sending certification TIFFs and
bundle files for more than one organism, please send one email per organism.
b. Name the TIFF and bundle files of your gel images as follows:
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 8 of 27
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
CODE: PNQ02
Effective Date:
04
26
10
TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two to
four letters of the file. The next 2 spaces will indicate the year the file was created. The next
3 spaces indicate the sequential number of the file submitted from your laboratory during a
calendar year. For example: GA09012.tif is the twelfth file submitted from the state of
Georgia during 2009.
Bundle: The bundle files are named in a similar manner as the TIFF files with the first two
to four letters of the file name indicating the unique identifier code for your laboratory. The
next 2 spaces indicate the year the bundle file was created, and the next 3 spaces indicate
the sequential bundle file number from each laboratory. For example, the eighth bundle file
submitted from the state of Georgia during 2009 would be named GA09008PN.bdl.
For each PulseNet pathogen, an individual may be certified for:
a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.
After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts
Appendix PNQ02-3
Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Shigella Cultures
Biological Safety Warning: Shigella strains are considered Level 2 biological agents by the U.S. Department of
Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry out laboratory
work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.
Note: Store the lyophilized cultures at 4oC in the dark until they are reconstituted.
Materials Needed:
Sturdy sterile forceps
1 ml pipetman
1 ml sterile pipet tips
1 ul sterile inoculating loop
Reagents Needed:
Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
Sterile grade reagent water
70% isopropyl alcohol
VERSION:
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STANDARD OPERATING PROCEDURE FOR
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Procedure for Reviving Cultures:
Day 1
1. Document the isolate number(s) and the date(s) lyophilized for your records. Wipe the aluminum cover and
outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber
stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial
with isopropyl alcohol before removing the stopper.
2. Re-suspend the lyophilized cells with 1ml of sterile grade reagent water. Allow to stand for a few minutes and/or
mix gently to produce a uniform suspension. With an inoculating loop, streak a small amount of this suspension onto
a blood agar plate (BAP) and incubate at 37ºC overnight.
Days 2 and 3
Check the BAP; if the culture appears pure, pick an isolated colony, and inoculate a fresh BAP for heavy growth;
incubate at 37oC overnight. Use the growth from this plate to make PFGE plugs of the standard strains. Before
making the plugs, transfer culture to fresh medium and incubate at 37oC overnight; this will ensure that the same
culture can be retested, if necessary.
After the strains have been reconstituted according to the above directions, streak each culture to agar plates, pick an
isolated colony, and subculture to another plate. Use the growth from the second plate to make the PFGE plugs.
Please let me know if the package does not arrive in satisfactory condition, or if the cultures are not viable. Freeze
(-70°C) or stock these strains according to your laboratory’s policy within 1 week after receiving them. Then,
your laboratory will have stock cultures of this PulseNet certification set for future use, including PulseNet
certification of additional personnel.
Biosafety Warning: Shigella species have a low infectious dose and are demonstrated hazards to laboratory
personnel; please use extreme caution and Biosafety Level 2 practices (at a minimum) when transferring and
handling Shigella strains. Work in a biological safety cabinet when handling large amounts of cells. Disinfect or
dispose of all plasticware and glassware that comes in contact with the cultures in a safe and appropriate manner.
The strain numbers of the Shigella sonnei cultures are as follows:
CDC90-01
CDC91-01
CDC94-01
CDC95-01
Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Escherichia coli,
non-typhoidal Salmonella serotypes and Shigella sonnei by Pulsed Field Gel Electrophoresis (PFGE)” Detailed
instructions for making the PFGE plugs can be found in the PulseNet QA/QC Manual PNL06. This document is
available in the QA/QC conference of the CDC Team Support or at www.cdc.gov/pulsenet/. If you can not access
the protocol, please request it by sending an E-mail to PFGE@CDC.GOV.
1.
2.
3.
4.
Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they
can be retested several times, if necessary.
Restrict one plug slice from each of the 4 test cultures (CDC90-01, CDC91-01, CDC94-01, and
CDC95-01) and 3 plug slices of H9812 (the PulseNet Universal Standard Strain) with XbaI (40-50
Units per plug slice) for 2 hours at 37ºC.
Restrict one plug slice from three of the test cultures (CDC90-01, CDC91-01, and CDC94-01)
with AvrII (BlnI; 25-30 Units/plug slice) for 2 hours at 37°C.
Load H9812 in Lanes 1, 6, 10 and the test strains as follows:
VERSION:
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STANDARD OPERATING PROCEDURE FOR
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Lane
1
2
3
4
5
6
7
8
9
10
Certification Strain
H9812
CDC90-01
CDC91-01
CDC94-01
CDC95-01
H9182
CDC90-01
CDC91-01
CDC94-01
H9182
CODE: PNQ02
Effective Date:
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Enzyme
XbaI
XbaI
XbaI
XbaI
XbaI
XbaI
BlnI
BlnI
BlnI
XbaI
Note: If you use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty
(Although the use of the ~0.5-mm wide 15-teeth combs [Bio-Rad, 170-4326] in the standard casting stand
[14 x 13-cm] is not recommended for routine PFGE analysis of test isolates, the use of the smaller comb
teeth will be allowed for certification.).
5.
Run the gel using the Shigella electrophoresis conditions. The run times listed in the protocol are
based on the equipment and reagents used at CDC. If the gels generated in your lab do not have
the lowest band in strain H9812 approximately 1-1.5 cm from the bottom of the gel, the run time
may have to be changed.
6.
Staining, de-staining, and gel documentation (imaging):
a. The gel image should fill the entire window of the imaging equipment (computer) screen
(without cutting off wells or lower bands). Individuals submitting certification TIFFs that do
not contain the wells or clearly show the bottom bands of the patterns that will automatically
be asked to rerun the gel and submit a new certification TIFF.
b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more
shadow bands (indicating incomplete restriction) should not be submitted for certification. To
eliminate incomplete restriction wash the plugs at least two more times with TE Buffer before
restriction is repeated. If the problem persists, repeat the restriction with more units of enzyme,
for a longer amount of time and/or with a different lot of enzyme..
7.
Bundle file creation
a.
In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC
strain number in the BioNumerics “Key” field. The bundle file should contain 4 database
entries as follows: 3 test strains restricted with XbaI and BlnI and one test strain restricted
only with XbaI. If there are more than 4 entries in the bundle file, one or more lanes have not
been linked properly.
b. Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines PND04
for instructions on how to create a PulseNet Bundle File). Bundles created using this icon are
“PulseNet bundles” and will contain only standardized PulseNet experiments and fields.
“PulseNet bundles” can be recognized by the “PN” that is automatically added to the file
name. If a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.
VERSION:
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8. Send the TIFF file and/or bundle (*.bdl) file of your gel images to CDC PulseNet at PFGE@CDC.GOV.
a. In the e-mail to CDC, include Shigella Certification in the subject line, so it can be forwarded
to the correct person. Include the isolate number and restriction enzyme used for each lane on
the gel in the body of the email. If you are sending certification TIFFs and bundle files for
more than one organism, please send one email per organism.
b. Name the TIFF and bundle files of your gel images as follows:
TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two to four
letters of the file. The next 2 spaces will indicate the year the file was created. The next 3 spaces
indicate the sequential number of the file submitted from your laboratory during a calendar year.
For example: GA09012.tif is the twelfth file submitted from the state of Georgia during 2009.
Bundle: The bundle files are named in a similar manner as the TIFF files with the first two to four
letters of the file name indicating the unique identifier code for your laboratory. The next 2 spaces
indicate the year the bundle file was created, and the next 3 spaces indicate the sequential bundle
file number from each laboratory. For example, the eighth bundle file submitted from the state of
Georgia during 2009 would be named GA09008PN.bdl.
For each PulseNet pathogen, an individual may be certified for:
a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.
After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts.
Appendix PNQ02-4
Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Listeria monocytogenes
Cultures
Biological Safety Warning: Listeria monocytogenes is considered a Level 2 biological agent by the U.S.
Department of Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry
out laboratory work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.
Note: Store the lyophilized culture at 4˚C in the dark until they are reconstituted.
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Materials Needed:
Sturdy sterile forceps
1 ml pipetman
1 ml sterile pipet tips
1 μl sterile inoculating loop
Reagents Needed:
Blood agar plate or equivalent media
Brain heart infusion agar plates
Sterile grade reagent water
70% isopropyl alcohol
Procedure for Reviving Cultures:
1.
Wipe the aluminum cover and outside of the vial with isopropyl alcohol. Using forceps, aseptically remove
the aluminum cover and rubber stopper from the vial containing the lyophilized culture. Wipe the outside
of the vial and stopper with alcohol after the metal cap is removed.
2.
Re-suspend the lyophilized cells with 1.0 mL of sterile reagent grade water. Allow to stand for a few
minutes and/or mix gently to produce a uniform suspension. Pipet 10µl of Listeria cell suspension onto a
blood agar plate (BAP) and with a 10µl loop streak for growth. Incubate at 37ºC overnight.
3.
Check the blood agar plate; if the culture appears pure, pick 2-3 representative colonies and inoculate a
fresh brain heart infusion agar plate for heavy growth; incubate at 37˚C for 18 to 24 hours. Use the growth
from the brain heart infusion agar plate to make agarose gel plugs as described in the Standardized
Molecular Subtyping of Foodborne Bacterial Pathogens by Pulsed-Field Gel Electrophoresis.
Listeria monocytogenes certification isolate plug slice loading position:
Lane number
1
2
3
4
5
6
7
8
9
10
Isolate number
S. Braenderup H9812
CDC-02-H8393
CDC-03-H8394
CDC-04-H8395
Lm control H2446
S. Braenderup H9812
CDC-02-H8393
CDC-03-H8394
CDC-04-H8395
S. Braenderup H9812
Restriction Enzyme
XbaI
AscI
AscI
AscI
AscI
XbaI
ApaI
ApaI
ApaI
XbaI
Recommended gel casting bed and comb: Standard casting stand, 14 x 13 cm frame/platform and 10 well comb, 14
cm wide, 1.5-mm thick.
After the strains have been reconstituted according to the above instructions, streak each culture to a Trypticase Soy
+ 5% Sheep’s blood agar plate (BAP) and allow them to incubate at 37°C for 24 hours to check for viability. Please
let me know if the shipment does not arrive in satisfactory condition, or if the cultures are not viable. Freeze (-70˚C)
or stock these strains immediately after reviving them according to your laboratory policy. The L. monocytogenes
strains in the revised set are:
VERSION:
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STANDARD OPERATING PROCEDURE FOR
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CDC#H8393
CDC#H8394 CDC#H8395
CODE: PNQ02
Effective Date:
04
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CDC#H2446 control strain
Please follow the Listeria monocytogenes standardized protocol (PNL04) for making the plugs and running the gel
for these isolates. Refer to the Standardized Laboratory Protocol for Molecular Subtyping of Foodborne Bacterial
Pathogens by PFGE (Section 5.3, Revised April, 2002).
Salmonella ser. Braenderup strain (H9812) is used as the reference standard. DNA of the H9812 strain must be
digested with XbaI to give the appropriate band pattern. Follow instructions in the E. coli O157:H7 Standardized
Laboratory protocol (PNL05) for making S. Braenderup plugs. If you do not have the S. Braenderup reference
standard you may request it from PFGE@CDC.GOV. The chart shown above lists the positions where plug slices
should be loaded on the gel.
Follow the instructions in steps 1-4 below for analysis, creating the bundle (*.bdl) file, and sending the TIFF(s)
and/or bundle file of your Listeria monocytogenes gel image(s) to CDC for certification.
1.
In the bundle file, do not include lanes from more than two gels. Enter the corresponding CDC strain
number in the BioNumerics “Key” field. The bundle file should contain 4 database entries as follows:
3 test strains restricted with AscI and ApaI and 1 test strain restricted only with AscI. If there are more
than 4 entries in the bundle file, one or more lanes have not been linked properly.
2.
Create the bundle file using the lightning bolt icon (see page 104 in the Appendix of the Dec. 2003
CDC BioNumerics manual). Bundles created using this icon are “PulseNet bundles” and will contain
only standardized PulseNet experiments and fields. “PulseNet bundles” can be recognized by the
“PN” that is automatically added to the file name. If a non-PulseNet bundle is submitted, you will
automatically be asked to resubmit.
3.
Send the TIFF file and/or bundle file of your gel images to CDC PulseNet at PFGE@CDC.GOV.
a.
In the e-mail to CDC, include the appropriate description of the gel (Listeria Certification) in
the subject line, so it can be forwarded to the correct person. Include the isolate number and
restriction enzyme used for each lane on the gel in the body of the e-mail or as an attachment.
If you are sending certification TIFFs and bundle files for more than one organism, please
send one e-mail per organism.
b.
Name the TIFF and bundle files of your gel images as follows:
TIFF: Use he unique identifier code that was assigned by CDC PulseNet for the first two,
three, or four letters of the file. The next 2 spaces will indicate the year the file was submitted.
The next 3 spaces indicate the sequential number of the file submitted from your laboratory
during a calendar year. For example: GA09012.tif is the twelfth file submitted from the state
of Georgia during 2009.
Bundle: The bundle files are named in a similar manner as the TIFF files with the first two,
three, or four letters of the file name indicating the unique identifier code for your laboratory.
The next 2 spaces indicate the year the bundle file was submitted, and the next 3 spaces
indicate the sequential bundle file number from each laboratory. For example, the eighth
bundle file submitted from the state of Georgia during 2009 would be named
GA09008PN.bdl.
c.
Please refer to Section 15 of the PulseNet PFGE Manual for additional information on naming
the TIFF and bundle files and submitting gel images to PulseNet.
4. For each PulseNet pathogen, an individual may be certified for:
VERSION:
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AUTHORIZED BY:
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CODE: PNQ02
STANDARD OPERATING PROCEDURE FOR
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a. TIFF image only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database)
c. Both TIFF and analysis
After the gel images are submitted, the certification file evaluator will analyze the gels and inform your laboratory of
your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the TIFF images
are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for analysis; if
the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID device (also
called a fob) and will be granted on-line access to the appropriate PulseNet national database. (Up to two SecurID
devices can be provided to a participating public health PulseNet laboratory. SecurID devices are issued to
individuals within a laboratory. The devices cannot be shared and must be returned to CDC if the certified individual
leaves his or her position in the laboratory.) If the submitted certification files are not satisfactory, the individual will
need to review the troubleshooting comments received from the evaluator and resubmit once results have improved.
If the submitter fails certification three times, the individual will not be allowed to submit again for six months.
Before resubmitting, the individual will be expected to work with CDC and/or their PulseNet Area Laboratory until
satisfactory results are achieved. This includes, but is not limited to troubleshooting and training in the PulseNet
PFGE protocols, BioNumerics and the PulseNet masterscripts.
Appendix PNQ02-5
Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Campylobacter jejuni
Cultures
Biological Safety Warning: Campylobacter jejuni strains are considered Level 2 biological agents by the U.S.
Department of Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry out
laboratory work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.
Note: Store the lyophilized cultures at 4oC in the dark until they are reconstituted.
Materials Needed:
Sturdy sterile forceps
1 ml pipetman
1 ml sterile pipet tips
1 ul sterile inoculating loop
Reagents Needed:
BHI agar + 5% Rabbit Blood or equivalent Campylobacter plating media
Sterile grade reagent water
70% isopropyl alcohol
Procedure for Reviving Cultures:
Day 1
1. Document the isolate number(s) and the date(s) lyophilized for your records. Wipe the aluminum cover and
outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber
stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial
with isopropyl alcohol before removing the stopper.
VERSION:
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2. Re-suspend the lyophilized cells with 250 μl of sterile grade reagent water. Allow to stand for a few minutes
and/or mix gently to produce a uniform suspension. Pipet 100µl of Campylobacter cell suspension onto a blood agar
plate (BAP) and with a 10µl loop streak for growth. Incubate at 37ºC for 48 hours.
Days 2 and 3
1. Check the plate; if the culture appears pure, pick an isolated colony, and inoculate a fresh plate for heavy growth;
incubate microaerobically for 24 hours at 37oC. Use the growth from this plate to make PFGE plugs of the C. jejuni
strains. Before making the plugs, transfer culture to fresh medium and incubate microaerobically at 37oC for 24
hours; this will ensure that the same culture can be retested, if necessary.
After the strains have been reconstituted according to the above directions, streak each culture to agar plates, pick an
isolated colony, and subculture to another plate. Use the growth from the second plate to make the PFGE plugs.
Please let me know if the package does not arrive in satisfactory condition, or if the cultures are not viable. Freeze
(-70°C) or stock these strains according to your laboratory’s policy within 1 week of receiving them. Then
your laboratory will have stock cultures of this PulseNet certification set for future use, including the
PulseNet certification of additional personnel.
The strain numbers of the Campylobacter cultures are as follows:
D424
D445
D2261
D2579
Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Campylobacter
jejuni by Pulsed Field Gel Electrophoresis (PFGE)” (PulseNet QA/QC Manual PNL03) for detailed instructions for
making the Campylobacter PFGE plugs and the “One-Day (24-28 h) Standardized Laboratory Protocol for
Molecular Subtyping of Escherichia coli, Salmonella serotypes and Shigella sonnei by Pulsed Field Gel
Electrophoresis (PFGE)” (PulseNet QA/QC Manual PNL05) to make the H9812 standard plugs.
Both sections are available in the PulseNet QA/QC Manual. This document is available in the QA/QC conference
of the CDC Team Support or at www.cdc.gov/pulsenet/. If you can not access the protocol, please request it by
sending an E-mail to PFGE@CDC.GOV.
1.
2.
3.
Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they
can be retested several times, if necessary.
Restrict one plug slice from the four test cultures (D424, D445, D2261, and D2579) with SmaI for
2 - 4 hours at 25°C and 2 plug slices of Salmonella ser. Braenderup H9812 standard with XbaI for
at least 2 hours at 37°C.
Load H9812 in Lanes 1 and 6 and the test strains as follows:
Lane
1
2
3
4
5
6
Certification Strain
H9812
D424
D445
D2261
D2579
H9812
Enzyme
XbaI
SmaI
SmaI
SmaI
SmaI
XbaI
Note: If you want to use combs with 15 teeth, load the plug slices in lanes 3-9 and leave the other lanes empty. (The
use of the ~0.5-mm wide 15-teeth comb [Bio-Rad, 170-4326] in the standard casting stand [14 x 13-cm] is not
recommended for routine PFGE analysis of test isolates, but its use will be allowed for certification.).
VERSION:
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AUTHORIZED BY:
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STANDARD OPERATING PROCEDURE FOR
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4.
Run the gel using the Campylobacter jejuni electrophoresis conditions. The run times listed in the
protocol are based on the equipment and reagents used at CDC. If the gels generated in your lab
do not have the lowest band in strain H9812 approximately 1-1.5 cm from the bottom of the gel,
the run time may have to be changed.
5.
Staining, de-staining, and gel documentation (imaging):
a. The gel image should fill the entire window of the imaging equipment (computer) screen
(without cutting off wells or lower bands). Individuals submitting certification TIFFs that do
not contain the wells or clearly show the bottom bands of the patterns that will automatically
be asked to rerun the gel and submit a new certification TIFF.
b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more
shadow bands (indicating incomplete restriction) should not be submitted for certification. To
eliminate incomplete restriction wash the plugs at least two more times with TE Buffer before
restriction is repeated. If the problem persist repeat the restriction with more units of enzyme,
for a longer amount of time and/or with a different lot of enzyme.
7.
Bundle file creation
a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC
strain number in the BioNumerics “Key” field. The bundle file should contain 4 database
entries (the 4 test strains) linked with SmaI.
b. Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines
PND04 for instructions on how to create a PulseNet Bundle File). Bundles created using this
icon are “PulseNet bundles” and will contain only standardized PulseNet experiments and
fields. “PulseNet bundles” can be recognized by the “PN” that is automatically added to the
file name. If a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.
8. Send the TIFF file and/or bundle (*.bdl) file of your gel images to the CDC Pulse Net Database
Administrator at PFGE@CDC.GOV within four weeks after receiving the strains.
a. In the email to CDC, include Campylobacter Certification in the Subject line, so it can be
forwarded to the correct person. Include the isolate number and restriction enzyme used for
each lane on the gel in the body of the email. If you are sending certification TIFFs and
bundle files for more than one organism, please send one email per organism.
b. Name the TIFF and bundle files of your gel images as follows:
TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two to
four letters of the file. The next 2 spaces will indicate the year the file was created. The next
3 spaces indicate the sequential number of the file submitted from your laboratory during a
calendar year. For example: GA09012.tif is the twelfth file submitted from the state of
Georgia during 2009. Bundle: The bundle files are named in a similar manner as the TIFF
files with the first two to four letters of the file name indicating the unique identifier code.
The next 2 spaces indicate the year the bundle file was created, and the next 3 spaces indicate
the sequential bundle file number from each laboratory. For example, the eighth bundle file
submitted from the state of Georgia during 2009 would be named GA09008PN.bdl.
For each PulseNet pathogen, an individual may be certified for:
a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.
After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
VERSION:
REPLACED BY:
AUTHORIZED BY:
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STANDARD OPERATING PROCEDURE FOR
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CODE: PNQ02
Effective Date:
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device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts.
Appendix PNQ02-6
Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Vibrio cholerae Cultures
This package contains lyophilized cultures of the four Vibrio cholerae strains that are used for PulseNet
Certification. After the strains have been reconstituted according to the enclosed directions, streak each culture to
agar plates, pick an isolated colony, and subculture to another plate. Use the growth from the second plate to make
the PFGE plugs. Please let us know if the package does not arrive in satisfactory condition, or if the cultures are not
viable. Freeze (-70°C) or stock these strains according to your laboratory’s policy using 24 – 48 h growth
within one week after reconstituting them. Then, your laboratory will have stock cultures of this PulseNet
certification set for future use, including the PulseNet certification of additional personnel.
The strain numbers of the Vibrio cultures are as follows:
CDC-100
CDC-104
CDC-105
CDC-106
Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Vibrio cholerae by
Pulsed Field Gel Electrophoresis (PFGE)” (PulseNet QA/QC Manual PNL06) for detailed instructions for making
the Vibrio PFGE plugs and the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of
Escherichia coli, Salmonella serotypes and Shigella sonnei by Pulsed Field Gel Electrophoresis (PFGE)” (PulseNet
QA/QC Manual PNL05) to make the H9812 standard plugs. Both sections are available in the PulseNet QA/QC
Manual. This document is available in the QA/QC conference on CDC Team or at www.cdc.gov/pulsenet/. If you
can not access the protocol, please request it by sending an E-mail to PFGE@CDC.GOV.
1. Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they can be
retested several times, if necessary.
2. Restrict one plug slice from the four test cultures (CDC-100, CDC-104, CDC-105, and CDC-106) with
SfiI for 4 hours at 50°C and 3 plug slices of Salmonella ser. Braenderup H9812 standard with XbaI for
at least 2 hours at 37°C.
3. Restrict one plug slice from the three test cultures (CDC-104, CDC-105, and CDC-106) with NotI for 4
hours at 37°C.
4. Load H9812 in Lanes 1, 6, 10 and the test strains as follows:
Lane
1
2
3
4
5
6
7
VERSION:
Certification Strain
H9812
CDC-100
CDC-104
CDC-105
CDC-106
H9182
CDC-104
REPLACED BY:
Enzyme
XbaI
SfiI
SfiI
SfiI
SfiI
XbaI
NotI
AUTHORIZED BY:
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STANDARD OPERATING PROCEDURE FOR
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8
9
10
CDC-105
CDC-106
H9182
CODE: PNQ02
Effective Date:
04
26
10
NotI
NotI
XbaI
Note: If you want to use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty.
(The use of the ~0.5-mm wide 15-teeth comb [Bio-Rad, 170-4326] in the standard casting stand [14 x 13-cm] is not
recommended for routine PFGE analysis of test isolates, but its use will be allowed for certification.).
5.
Run the gel using the V. cholerae electrophoresis conditions. The run times listed in the protocol
are based on the equipment and reagents used at CDC. If the gels generated in your lab do not
have the lowest band in strain H9812 approximately 1-1.5 cm from the bottom of the gel, the run
time may have to be changed.
6.
Staining, de-staining, and gel documentation (imaging):
a. The gel image should fill the entire window of the imaging equipment (computer) screen
(without cutting off wells or lower bands). Individuals submitting certification TIFFs that do
not contain the wells or clearly show the bottom bands of the patterns that will automatically
be asked to rerun the gel and submit a new certification TIFF.
b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more
shadow bands (indicating incomplete restriction) should not be submitted for certification. To
eliminate incomplete restriction wash the plugs at least two more times with TE Buffer before
restriction is repeated. If the problem persist repeat the restriction with more units of enzyme,
for a longer amount of time and/or with a different lot of enzyme.
6. Bundle file creation
a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC
strain number in the BioNumerics “Key” field. The bundle file should contain 4 database entries
as follows: 3 test strains restricted with SfiI and NotI and one test strain restricted only with SfiI.
If there are more than 4 entries in the bundle file, one or more lanes have not been linked
properly.
b.
Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines
PND04 for instructions on how to create a PulseNet Bundle File). Bundles created using this
icon are “PulseNet bundles” and will contain only standardized PulseNet experiments and
fields. “PulseNet bundles” can be recognized by the “PN” that is automatically added to the file
name. If a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.
7. Send the TIFF file and/or bundle (*.bdl) file of your gel image to CDC at PFGE@cdc.gov within four
weeks after receiving the strains.
a. In the email to CDC, include Vcholerae Certification in the Subject line, so it can be
forwarded to the correct person. Include the isolate number and restriction enzyme used for each
lane on the gel in the body of the email. If you are sending certification TIFFs and bundle files for
more than one organism, please send one email per organism.
b. Name the TIFF and bundle files of your gel images as follows:
TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two to
four letters of the file. The next 2 spaces will indicate the year the file was created. The
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 19 of 27
STANDARD OPERATING PROCEDURE FOR
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CODE: PNQ02
Effective Date:
04
26
10
next 3 spaces indicate the sequential number of the file submitted from your laboratory
during a calendar year. For example: GA09012.tif is the twelfth file submitted from the
Georgia Public Health Laboratory during 2009.
Bundle: The bundle files are named in a similar manner as the TIFF files with the first two
to four letters of the file name indicating the unique identifier code. The next 2 spaces
indicate the year the bundle file was created, and the next 3 spaces indicate the sequential
bundle file number from each laboratory. For example, the eighth bundle file submitted
from the Georgia Public Health Laboratory during 2009 would be named GA09008PN.bdl.
Remember, “PN” is automatically added to the file name.
For each PulseNet pathogen, an individual may be certified for:
a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.
After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory.
If the TIFF images or bundle files are not satisfactory, the submitter will need to review the troubleshooting
comments received from the evaluator and resubmit once results have improved. If the submitter fails certification
three times, the individual will not be allowed to submit again for six months. Before resubmitting, the individual
will be expected to work with CDC and/or their PulseNet Area Laboratory until satisfactory results are achieved.
This includes, but is not limited to, troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and
the PulseNet Master Scripts.
.
Appendix PNQ02-7
Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) Vibrio parahaemolyticus
Cultures
Biological Safety Warning: Vibrio parahaemolyticus strains are considered Level 2 biological agents by the U.S.
Department of Health and Human Services. Use appropriate precautions when handling the vial or culture. Carry
out laboratory work in a biological safety cabinet when applicable to ensure aseptic conditions and personal safety.
Note: Store the lyophilized cultures at 4°C in the dark until they are reconstituted.
Materials Needed:
Sterile sturdy forceps
1 ml pipetman
1 ml sterile pipet tips
1 ul sterile inoculating loop
Reagents Needed:
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 20 of 27
STANDARD OPERATING PROCEDURE FOR
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CODE: PNQ02
Effective Date:
04
26
10
Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
Sterile grade reagent water or Trypticase Soy Broth (TSB)
70% isopropyl alcohol
Procedure for Reviving Cultures:
Day 1
1. Document the isolate number (s) and the date(s) lyophilized for your records. Wipe the aluminum cover and
outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the aluminum cover and rubber
stopper from the vial containing the lyophilized culture. Wipe the outside of the rubber stopper and neck of the vial
with isopropyl alcohol before removing the stopper.
2. Re-suspend the lyophilized cells with 1.0 ml of sterile grade reagent water. Allow to stand for a few minutes
and/or mix gently to produce a uniform suspension. Pipet 100µl of cell suspension onto a blood agar plate (BAP)
and with a 10µl loop streak for growth. Incubate at 37ºC overnight.
Days 2 and 3
1. Check the BAP; if the culture appears pure, pick an isolated colony, and inoculate a fresh BAP for heavy growth;
incubate at 37°C overnight. Use the growth from this plate to make PFGE plugs of the standard strains. Before
making the plugs, transfer culture to fresh medium and incubate at 37°C overnight; this will ensure that the same
culture can be retested, if necessary.
After the strains have been reconstituted according to the above directions, streak each culture to agar plates, pick an
isolated colony, and subculture to another plate. Use the growth from the second plate to make the PFGE plugs.
Please let us know if the package does not arrive in satisfactory condition, or if the cultures are not viable. Freeze (70°C) or stock these strains according to your laboratory’s policy using 24 – 48 h growth within one week
after reconstituting them. Then, your laboratory will have stock cultures of this PulseNet certification set for
future use, including the PulseNet certification of additional personnel.
The strain numbers of the Vibrio cultures are as follows:
CDC#200-07
CDC#201-07
CDC#202-07
CDC#203-07
Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “One-Day (24-28 h) Standardized Laboratory Protocol for Molecular Subtyping of Vibrio
paraheamolyticus by Pulsed Field Gel Electrophoresis (PFGE)” (PulseNet QA/QC Manual PNL22) for detailed
instructions for making the Vibrio PFGE plugs and the “One-Day (24-28 h) Standardized Laboratory Protocol for
Molecular Subtyping of Escherichia coli, Salmonella serotypes and Shigella sonnei by Pulsed Field Gel
Electrophoresis (PFGE)” (PulseNet QA/QC Manual PNL05) to make the H9812 standard plugs. Both sections are
available in the PulseNet QA/QC Manual. This document is available in the QA/QC conference on CDC Team or at
www.cdc.gov/pulsenet/. If you can not access the protocol, please request it by sending an E-mail to
PFGE@CDC.GOV.
1. Make 2-3 (disposable plug molds) or two (reusable plug molds) plugs of each test strain so they
can be retested several times, if necessary.
2. Restrict one plug slice from the four test cultures (CDC#200-07, CDC#201-07, CDC#202-07,
CDC#203-07) with SfiI for 4 hours at 50°C and 3 plug slices of Salmonella ser. Braenderup
H9812 standard with XbaI for at least 2 hours at 37°C.
3. Restrict one plug slice from three of the three test cultures (CDC#200-07, CDC#201-07, and
CDC#202-07) with NotI for 4 hours at 37°C.
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 21 of 27
STANDARD OPERATING PROCEDURE FOR
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4.
CODE: PNQ02
Effective Date:
04
26
10
Load H9812 in Lanes 1, 6, 10 and the test strains as follows:
Lane
1
2
3
4
5
6
7
8
9
10
Certification Strain
H9812
CDC#200-07
CDC#201-07
CDC#202-07
CDC#203-07
H9182
CDC#200-07
CDC#201-07
CDC#202-07
H9182
Enzyme
XbaI
SfiI
SfiI
SfiI
SfiI
XbaI
NotI
NotI
NotI
XbaI
Note: If you want to use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty.
(The use of the ~0.5-mm wide 15-teeth comb [Bio-Rad, 170-4326] in the standard casting stand [14 x 13-cm] is not
recommended for routine PFGE analysis of test isolates, but its use will be allowed for certification.).
5. Run the gel using the V. paraheamolyticus electrophoresis conditions. The run times listed in the
protocol are based on the equipment and reagents used at CDC. If the gels generated in your lab do not
have the lowest band in strain H9812 approximately 1-1.5 cm from the bottom of the gel, the run time
may have to be changed.
6. Staining, de-staining, and gel documentation (imaging):
a. The gel image should fill the entire window of the imaging equipment (computer) screen (without
cutting off wells or lower bands). Individuals submitting certification TIFFs that do not contain the
wells or clearly show the bottom bands of the patterns that will automatically be asked to rerun the
gel and submit a new certification TIFF.
b. TIFFs with one lane that contains many shadow bands or multiple lanes with one or more shadow
bands (indicating incomplete restriction) should not be submitted for certification. To eliminate
incomplete restriction wash the plugs at least two more times with TE Buffer before restriction is
repeated. If the problem persist repeat the restriction with more units of enzyme, for a longer
amount of time and/or with a different lot of enzyme.
7. Bundle file creation
a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC strain
number in the BioNumerics “Key” field. The bundle file should contain 4 database entries as
follows: 3 test strains restricted with SfiI and NotI and one test strain restricted only with SfiI. If
there are more than 4 entries in the bundle file, one or more lanes have not been linked properly.
b. Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines PND04
for instructions on how to create a PulseNet Bundle File.). Bundles created using this icon are
“PulseNet bundles” and will contain only standardized PulseNet experiments and fields.
“PulseNet bundles” can be recognized by the “PN” that is automatically added to the file name. If
a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.
8. Send the TIFF file and/or bundle (*.bdl) file of your gel image to CDC at PFGE@cdc.gov within four
weeks after receiving the strains.
a. In the email to CDC, include Vparahaem Certification in the Subject line, so it can be forwarded
to the correct person. Include the isolate number and restriction enzyme used for each lane on the
gel in the body of the email. If you are sending certification TIFFs and bundle files for more than
one organism, please send one email per organism.
b. Name the TIFF and bundle files of your gel images as follows:
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 22 of 27
CODE: PNQ02
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
Effective Date:
04
26
10
TIFF: Use the unique laboratory identifier code that was assigned by CDC PulseNet for the first
two to four letters of the file. The next 2 spaces will indicate the year the file was created. The next
3 spaces indicate the sequential number of the file submitted from your laboratory during a calendar
year. For example: GA09012.tif is the twelfth file submitted from the Georgia Public Health
Laboratory during 2009.
Bundle: The bundle files are named in a similar manner as the TIFF files with the first two to four
letters of the file name indicating the unique identifier code. The next 2 spaces indicate the year the
bundle file was created, and the next 3 spaces indicate the sequential bundle file number from each
laboratory. For example, the eighth bundle file submitted from the Georgia Public Health
Laboratory during 2009 would be named GA09008PN.bdl. Remember, “PN” is automatically
added to the file name.
For each PulseNet pathogen, an individual may be certified for:
a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.
After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory. If the TIFF images or bundle files are not
satisfactory, the submitter will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet Master Scripts.
Appendix PNQ02-8
Analysis Certification Protocol for E. coli Non O157 (STEC)
PulseNet participants may become analysis-certified for E. coli Non O157 (STEC) by analyzing a TIFF
sent by CDC (KC09104.tif) and creating a PulseNet bundle file. There must be at least one person in your
laboratory that is currently TIFF-certified in E. coli O157:H7 before anyone else from your laboratory
becomes analysis-certified for E. coli Non O157.
1. Lane Information for KC09104.tif.
Note 1: If more than one person in your laboratory will be submitting this certification set, you may wish to
rename the TIFF using your initials (i.e. JKA09104.tif or KC09104JKA.tif) before you import it into your
local database (otherwise the TIFF will be overwritten each time it is imported by a different person).
Note 2: When assigning reference markers in step 3 (normalization) of analysis place the marker in the
middle of the top band (668.9 Kb) if you resolve a single band and place the marker on the lower of the two
bands if you resolve a doublet.
Lane
1
VERSION:
Certification Strain
H9812
REPLACED BY:
Enzyme
XbaI
AUTHORIZED BY:
Page 23 of 27
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
2
3
4
5
6
7
8
9
10
3074-01
3508-03
K5159
K4947
H9182
3074-01
3508-03
K5159
H9182
CODE: PNQ02
Effective Date:
04
26
10
XbaI
XbaI
XbaI
XbaI
XbaI
BlnI
BlnI
BlnI
XbaI
2. Bundle File Creation
a. In the bundle file enter the corresponding CDC strain number in the BioNumerics “Key” field. If
there are more than 7 entries in the bundle file, one or more lanes have not been linked properly.
b. Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines PND04 for
instructions on how to create a PulseNet Bundle File). Bundles created using this icon are
“PulseNet bundles” and will contain only standardized PulseNet experiments and fields. “PulseNet
bundles” can be recognized by the “PN” that is automatically added to the file name. If a nonPulseNet bundle is submitted, you will automatically be asked to resubmit.
3. Send the bundle (*.bdl) file of your gel image to CDC at PFGE@cdc.gov.
a. In the email to CDC, include E. coli Non O157 Certification in the Subject line, so it will be
directed to the correct person. If you are submitting certification TIFFs and bundle files for more
than one organism, please send one email per organism.
b. Name the bundle file containing your analysis as follows:
Bundle files are named in a similar manner as TIFF files with the first two to four letters of the file
name indicating your unique laboratory identifier code. The next 2 spaces indicate the year the
bundle file was created, and the next 3 spaces indicate the sequential bundle file number from each
laboratory. For example, the eighth bundle file submitted from the Georgia Public Health
Laboratory during 2009 would be named GA09008PN.bdl. Remember, “PN” is automatically
added to the file name.
After your analysis bundle file is submitted, the PulseNet certification evaluator will analyze your submission and
inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the
files. If the bundle file is satisfactory, the person who submitted the bundle file will be issued a SecurID device (also
called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID devices are
issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if the certified
individual leaves his or her position in the laboratory. If the bundle file is not satisfactory, the submitter will need to
review the troubleshooting comments received from the evaluator and resubmit once results have improved. If the
submitter fails certification three times, the individual will not be allowed to submit again for six months. Before
resubmitting, the individual will be expected to work with CDC and/or their PulseNet Area Laboratory until
satisfactory results are achieved. This includes, but is not limited to, troubleshooting and training in the PulseNet
protocols, BioNumerics and the PulseNet Master Scripts.
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 24 of 27
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
CODE: PNQ02
Effective Date:
04
26
10
Appendix PNQ02-9
Analysis Certification Protocol for C. botulinum
This package contains cultures of four Clostridium botulinum strains used for PulseNet Certification. Since this is a
Select Agent transfer, your Responsible Official (RO) must complete Section 3 of APHIS/CDC Form 2 and send a
copy to APHIS/CDC within two business days of receipt. If the package has been damaged to the extent that a
release of select agent may have occurred, your laboratory’s RO must immediately report to APHIS/CDC. Please
refer to National Select Agent Registry website (http://www.selectagents.gov/TransferForm.html) for additional
information.
Stock these four C. botulinum strains according to your laboratory’s policy within one week after receiving them.
Then, your laboratory will have stock cultures of this PulseNet certification set for future use, including the PulseNet
certification of additional personnel.
The strain numbers of the C. botulinum cultures are as follows:
CDC5328 CDC44575
CDC37456
CDC55707
Please follow these supplemental instructions for making the PFGE plugs and running the gels for these strains.
Refer to the “Five Day Standardized Laboratory Protocol for Molecular Subtyping of Clostridium botulinum
by Pulsed Field Gel Electrophoresis (PFGE). This document is available as PNL25 in the QA/QC Manual
conference on CDC Team or at www.cdc.gov/pulsenet/. If you cannot access the protocol, please request it by
sending an E-mail to PFGE@CDC.GOV.
1.
Streak each test culture for colony isolation onto EYA plates. Record isolate number and date on each
plate. Incubate under anaerobic conditions at 37°C (± 2) for two days.
2.
If the culture appears pure, pick an isolated colony and streak for colony isolation onto ANA-BAP plates.
Incubate under anaerobic conditions overnight at 37°C ± 2°C.
3.
Make two or three disposable plug molds plugs, or two reusable plug molds plugs of each test strain so they
can be retested several times, if necessary.
4.
Restrict three plug slices of Salmonella ser. Braenderup H9812 (the PulseNet Universal Standard Strain)
with XbaI (60 Units/plug slice) for 2 hours at 37°C.
5.
Restrict one plug slice from the four test cultures (CDC5328, CDC44575, CDC37456, and CDC55707)
with SmaI (50 Units/plug slice) for 4 hours at 25°C.
6.
Restrict one plug slice from three of the test cultures (CDC5328, CDC44575, and CDC37456) with XhoI
(100 Units/plug slice) for 3 hours at 37oC.
7.
Load H9812 and the test strains as follows:
Lane
1
2
3
4
5
VERSION:
Certification Strain
H9812
CDC5328
CDC44575
CDC37456
H9182
REPLACED BY:
Enzyme
XbaI
SmaI
SmaI
SmaI
XbaI
AUTHORIZED BY:
Page 25 of 27
STANDARD OPERATING PROCEDURE FOR
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6
7
8
9
10
CDC5328
CDC44575
CDC37456
CDC55707
H9182
CODE: PNQ02
Effective Date:
04
26
10
XhoI
XhoI
XhoI
SmaI
XbaI
Note: If you use combs with 15 teeth, load the plug slices in lanes 2-11 and leave the other lanes empty
(Although the use of the ~0.5-mm wide 15-teeth combs [Bio-Rad, 170-4326] in the standard casting stand
[14 x 13-cm] is not recommended for routine PFGE analysis of test isolates, the use of the smaller comb
teeth will be allowed for certification.).
8.
Run the gel using the C. botulinum electrophoresis conditions. These run times are based on the equipment
and reagents used at CDC. If the gels generated in your lab do not have the lowest band in strain H9812
approximately 1-1.5 cm from the bottom of the gel, the run time may have to be changed.
9.
Staining, de-staining, and gel documentation (imaging):
a.
The gel image should fill the entire window of the imaging equipment (computer) screen (without
cutting off wells or the bottom of the gel). An individual submitting a certification TIFF with wells
cut off or bottom bands that are not visible will automatically be asked to rerun the gel and submit
a new certification TIFF.
a.
TIFFs with one lane that contains many shadow bands or multiple lanes with one or more shadow
bands (indicating incomplete restriction) should not be submitted for certification. To eliminate
incomplete restriction wash the plugs at least two more times with TE Buffer before restriction is
repeated. If the problem persists, repeat the restriction with more units of enzyme, for a longer
amount of time and/or with a different lot of enzyme.
10. Bundle file creation
a. In the bundle file, do not include lanes from more than 2 gels. Enter the corresponding CDC strain
number in the BioNumerics “Key” field. The bundle file should contain 4 database entries as
follows: 3 test strains restricted with SmaI and XhoI and one test strain restricted only with SmaI.
If there are more than 4 entries in the bundle file, one or more lanes have not been linked properly.
b
Create the bundle file using the lightning bolt icon (refer to the Gel Analysis Guidelines PND04
for instructions on how to create a PulseNet Bundle File). Bundles created using this icon are
“PulseNet bundles” and will contain only standardized PulseNet experiments and fields.
“PulseNet bundles” can be recognized by the “PN” that is automatically added to the file name. If
a non-PulseNet bundle is submitted, you will automatically be asked to resubmit.
11. Send the TIFF file and/or bundle (*.bdl) file of your gel images to CDC PulseNet at PFGE@CDC.GOV
within four weeks after receiving the strains.
a.
In the email to CDC, include C. botulinum Certification in the Subject line, so it can be
forwarded to the correct person. Include the isolate number and restriction enzyme used for each
lane on the gel in the body of the email.
b.
Name the TIFF and bundle files of your gel images as follows:
TIFF: Use the unique identifier code that was assigned by CDC PulseNet for the first two or three
letters of the file. The next 2 spaces will indicate the year the file was submitted. The next 3 spaces
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 26 of 27
CODE: PNQ02
STANDARD OPERATING PROCEDURE FOR
CERTIFICATION OF PULSENET PERSONNEL
Effective Date:
04
26
10
indicate the sequential number of the file submitted from your laboratory during a calendar year. For
example: GA10012.tif is the twelfth file submitted from the state of Georgia during 2010.
Bundle: The bundle files are named in a similar manner as the TIFF files with the first 2 or three letters
of the file name indicating the unique identifier code for your laboratory. The next 2 spaces indicate
the year the bundle file was submitted, and the next 3 spaces indicate the sequential bundle file number
from each laboratory. For example, the eighth bundle file submitted from the state of Georgia during
2010 would be named GA10008PN.bdl.
For each PulseNet pathogen, an individual may be certified for:
a. Gels only (i.e., laboratory methods for PFGE and image acquisition)
b. Analysis only (i.e., BioNumerics analysis and on-line access to the database), or
c. Both gels and analysis.
After the gel images are submitted, the PulseNet certification file evaluator will analyze the gels and inform your
laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of receiving the files. If the
TIFF images are satisfactory, the person who submitted the TIFF will be eligible to send gel images to PulseNet for
analysis; if the bundle files are satisfactory, the person who submitted the bundle files will be issued a SecurID
device (also called a fob) and will be granted on-line access to the appropriate PulseNet national database. SecurID
devices are issued to individuals within a laboratory. The devices cannot be shared and must be returned to CDC if
the certified individual leaves his or her position in the laboratory. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the evaluator and
resubmit once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work with CDC
and/or their PulseNet Area Laboratory until satisfactory results are achieved. This includes, but is not limited to,
troubleshooting and training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts.
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STANDARD OPERATING PROCEDURE FOR
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1.
PURPOSE: To describe the procedure for evaluating certification files sent by PulseNet participating
laboratories. This is part of the PulseNet QA/QC program.
2.
SCOPE: This procedure applies to PulseNet personnel who evaluate certification files and certify individuals
for gels (laboratory methods for PFGE and image acquisition) and/or BioNumerics gel analysis. It also applies
to individuals who review certification reports.
3.
DEFINITIONS/TERMS:
3.1 Certification files: TIFF and/or bundle files submitted by PulseNet participating laboratories for certification
evaluation.
3.2 QA/QC: Quality Assurance/Quality Control
3.3 PFGE: Pulsed-field Gel Electrophoresis
3.4 BioNumerics: Gel analysis software used by PulseNet, developed by Applied Maths, Belgium
3.5 SOP: Standard Operating Procedure
3.6 TIFF: Tagged Image File Format, a file of a gel image that can be analyzed in BioNumerics
3.7 Certification file evaluator: An individual who evaluates certification files
3.8 TIFF quality: The grading of the appearance and ease of analysis of a TIFF according to the PulseNet TIFF
Grading Guidelines. This is a main component of the evaluation of a TIFF submitted for certification
3.9 Gel analysis assessment: The grading of the whole analysis of a TIFF, including gel and lane definition,
normalization, and band marking, according to the PulseNet Gel Analysis Guidelines. This is a main
component of the evaluation of a bundle file submitted for certification
3.10 Bundle file: A file with a .bdl extension that is produced in BioNumerics and contains the analysis of at least
one lane of a gel image
3.11 CDC: Centers for Disease Control and Prevention
3.12 Certification file reviewer: An individual who reviews and signs off on the certification reports submitted by
the certification file evaluator
3.13 Comparison TIFFs: One or more TIFFs run by CDC for a specific pathogen for use in comparing PFGE
patterns and band resolution against submitted certification TIFFs. Comparison TIFFs can also be a group of
certification TIFFs submitted by several laboratories to monitor PFGE patterns and band resolution over
several submitting laboratories. The latter is most easily accomplished through a saved list in BioNumerics
3.14 Comparison list: A list of analyzed lanes from comparison TIFFs that is saved in BioNumerics and used to
compare to the analysis of the submitted TIFF by the certification file evaluator and the analysis of the
submitter in the certification bundle file
3.15 Gel certified: Formerly “TIFF certified,” an individual or laboratory that is certified in laboratory methods for
PFGE and image acquisition
3.16 Analysis certified: An individual who is certified in BioNumerics gel analysis
4.
RESPONSIBILITIES
4.1 Individuals performing PulseNet-related work (i.e., preparing PFGE gels and/or analyzing TIFF images) must
submit certification file(s) and have them evaluated before being allowed to submit TIFF images and
BioNumerics analyses directly to the PulseNet national databases. See PNQ02 for information on how to
request certification sets.
4.2 Individuals evaluating certification files (evaluators) must:
4.2.1
Assess the TIFF quality and the ease of analysis of the TIFFs submitted for certification
and assess the gel analysis, including band marking, of bundle files submitted for certification. All
files submitted should be evaluated based on the current PulseNet standards of TIFF quality and gel
analysis.
4.2.2
Evaluate submitted files and submit a written report, using the existing templates, to
CDC reviewers within the time frame written in the certification instructions sent to the participating
laboratories with the certification strain sets.
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4.2.4
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Modify reports based on CDC reviewers’ comments, if necessary. Resubmit reports to
CDC as soon as possible.
Keep electronic files in an organized folder system and keep hard copies of TIFFs,
worksheets, reports, and any analyses (e.g., dendrograms, lane-to-lane comparisons) generated in files
organized by laboratory.
4.3 Individuals reviewing certification reports submitted by the evaluator (reviewers) must:
4.3.1
Review submitted reports and their associated cover letters and certificates within the
time frame written in the certification instructions sent to the participating laboratories with the
certification strain sets.
4.3.2
Submit signed reports to the next reviewer or to the Database Team Leader (Kelley
Hise).
5. PROCEDURE
5.1 TIFF and/or bundle files are received via email to the PFGE inbox are moved into the organism pending
subfolder within the QA/QC folder. The person assigned to evaluate that organisms’ certification file will
retrieve the submissions from this location.
5.2 The email is printed and the attached files are saved to a folder on the hard drive (or network drive) that
indicates which pathogen, which laboratory submitted the files, and which person in the laboratory submitted
the files (P:\QAQC\Certification\Certification-Final Reports\organism\lab ID-initials of submitter).
5.3 The file names and the date received are recorded in the appropriate Excel sheet (e.g., Salmonella Certification
Status_Steward.xls). The Excel sheets are saved to P:\QAQC\Certification\Certification Tables and updated
regularly.
5.4 When ready to analyze, open the TIFF in an image program. Invert to black bands on a white background, if not
submitted this way. Print out the TIFF picture, filling the whole 8 ½ x 11” sheet with the image, if possible.
5.5 Grade the TIFF visually; using the TIFF Grading Guidelines and also compare PFGE patterns to hard copies of
comparison TIFF(s).
5.5.1
If the TIFF is not passable (e.g., too much debris, bands not resolved well enough for
analysis, one or more PFGE patterns are not correct), email the sender to repeat the TIFF. Give
specific instructions about how to improve the next gel. Write on the printed email that you requested a
repeat submission and file in your working folder. Record repeat submission request on the appropriate
Excel sheet.
5.5.2
If the TIFF is passable, perform analysis as outlined in 5.6 below.
5.6 Analysis of certification TIFFs
5.6.1
Fill out the top third of a log-in and analysis sheet (login & analysis_worksheet.doc),
recording information about the current certification files and about any previous certification files
submitted but repeated.
5.6.2
In the middle third of the log-in and analysis sheet, record information about the gel
quality. Use the PulseNet TIFF Grading Guidelines, circling a rating for each category listed (4 being
the highest rating and 1 being the lowest). To the right of each category, record specific information
pertaining to that rating (e.g., Bands: clear & distinct? 2 “Bands very fuzzy and difficult to analyze in
some places”.)
5.6.3
In the bottom third of the log-in and analysis sheet, record information about the
following:
5.6.3.1 Standards – Are the standards in the correct lanes? Are the patterns correct and
distinguishable according to the current PulseNet standards and comparison TIFF(s)?
5.6.3.2 TIFF: “By eye”-match comparison gel? – Are the PFGE patterns of the test lanes
correct and distinguishable according to the current PulseNet standards and comparison
TIFF(s)?
5.6.4
Analyze the TIFF in BioNumerics. Analyze all 10 lanes of the TIFF (standard and test
lanes). Code each “key” entry with the Laboratory ID, the submitter’s initials, and the strain number
(e.g., CDC-KH-H9812).
5.6.4.1 If no bundle file was submitted, perform a dendrogram match with the comparison
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5.6.6
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list (the Salmonella list is named “2003_cert”; the E. coli list is named “2003comp” and the
Listeria list is named “Compcert”) using PulseNet standard settings. Proceed to step 5.6.6 of
this document.
5.6.4.2 If a bundle file was submitted, go to step 5.6.5 below.
Bundle file analysis
5.6.5.1 Highlight the existing bundle file in BioNumerics and check the information before
opening. Note whether there are non-PulseNet standardized fields and/or experiments in the
bundle file. Make sure all entries are in the bundle file and linked correctly. If the lanes are
linked incorrectly or if there are no standards in the bundle file, send an email to request a
repeat submission, noting any other changes that need to be made in the repeat submission.
5.6.5.2 Open the bundle file and select the lanes (noting if the lanes are linked incorrectly).
5.6.5.3 Using PulseNet standard settings, perform a dendrogram match of the comparison
list, the bundle file analysis, and the analysis of the TIFF by the evaluator. Proceed to step
5.6.6 of this document.
Analysis of TIFF and/or bundle file once dendrogram is generated in BioNumerics.
5.6.6.1 Print out a hard copy of the dendrogram and go through each isolate match, circling
where the discrepancies are and looking at the submitted TIFF to see if that TIFF has a
different band resolution than the comparison.
5.6.6.2 Underneath the dendrogram, record information for each isolate with a discrepancy.
For example:
5.6.6.2.1 “A single band is resolved at ~235 kb. Some labs are resolving a doublet at this position.”
If some laboratories are resolving a particular area as a single band and some as a doublet,
this is passable. If all labs are resolving a particular area as a doublet and the certification
TIFF shows a thick single band, the TIFF should be repeated (do not pass the TIFF or
bundle file for certification), especially if this occurs in more than one place on the TIFF.
5.6.6.2.2 “A doublet is marked on the bundle file at ~400 kb that appears as a single band on the
TIFF.” – If a laboratory “over-marks” several areas of the TIFF, request a repeat bundle
file.
5.6.6.2.3 “A doublet is marked on the bundle file at ~170 kb. It is difficult to distinguish a doublet
from a single band at ~170 kb on the TIFF.” – Use this comment when bands are fuzzy
and difficult to distinguish in a certain area and the submitter has marked the area on the
bundle differently than the certification evaluator has marked it. Ask for a repeat
submission if the fuzzy bands occur over more than several places on the TIFF and the
evaluator feels that better band resolution would dramatically improve analysis.
5.6.6.2.4 “A band is marked in error at ~55 kb where no band appears on the TIFF”. – If a
laboratory marks stray bands in more than one place, request a repeat bundle file.
5.6.6.2.5 Standard lanes with bands not marked – ask for a repeat bundle file if standard bands are
not marked at all. If band(s) at ~170 kb (the bands not used for normalization) are not
marked, record standard comment about marking appropriate bands for normalization but
mark all bands during band finding.
5.6.6.3 Note any bands marked on the bundle file that consistently appear above or below
the bands marked by the evaluator. This could be due to incorrect normalization by the
submitter. Check to make sure the standard the submitter used is correct. Ask for a repeat
bundle submission if the normalization is incorrect. The submitter can be gel certified, but
not analysis certified until a new bundle file is evaluated and passed.
5.6.6.4 In the bottom third of the log-in and analysis worksheet, record information about
the dendrogram analyses:
5.6.6.4.1
TIFF: Analyzed TIFF matches comparisons? - How well does the analysis of the
submitted TIFF by the certification file evaluator match the comparison list of TIFF(s)
analysis? This should include information about the band resolution and ease of analysis
of the submitted TIFF. A note of “See dendrogram notes” in this box on the worksheet
may be sufficient.
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5.6.6.4.2
Bundle: Analyzed TIFF matches bundle? – How well does the submitter’s bundle file
analysis match the analysis of the submitted TIFF by the evaluator? This should include
information about inaccurate normalization or band marking. A note of “See dendrogram
notes” in this box on the worksheet may be sufficient. If a bundle file was not submitted,
no information should be recorded in this box.
5.6.6.4.3 Bundle: Bundle patterns match comparison patterns? – How well does the submitter’s
bundle file analysis match the patterns generated by CDC and other laboratories. The
CDC patterns are those within the list used to compare to the bundle file in the
dendrogram generated. If necessary, the certification file evaluator may perform a
dendrogram match using submissions from other laboratories to see if doublets or single
bands are being submitted in a certain area of a particular lane. If a bundle file was not
submitted, no information should be recorded in this box.
5.6.6.4.4 Report Comments – Use this box for recording information such as if the band resolution
is much better or much worse than other submissions.
5.7 After analysis, save the report template for that pathogen to the folder where the certification files
were saved (P:\QAQC\Certification\Certification-Final Reports\organism\lab ID-initials of submitter). Save
the report template using the submitter’s name (e.g., Salm 2003 cert report_KH.doc).
5.8 Complete the report
5.8.1
Type in the comments recorded on the dendrogram for each isolate with a discrepancy or other
comment.
5.8.2
Type in suggestions for improvement and additional comments at the bottom of the report. Use the
Standard Comments document (Appendix PNQ04-1) for wording and troubleshooting tips.
5.8.3
Type in information about previous submissions and the use of non-PulseNet standard fields and
experiments in the bundle file at the bottom of the comments section. See Standard Comments
document for wording.
5.8.4
Make sure the circles around the “TIFF satisfactory” and “bundle satisfactory” are correct for each
report.
5.8.5
Print out two copies of the report – one to send to CDC and one for the evaluator’s files.
5.9 After completing the report, save the cover letter template for that pathogen to the folder where the certification
files and report were saved (P:\QAQC\Certification\Certification-Final Reports\organism\lab ID-initials of
submitter). Save the cover letter template using the submitter’s name (e.g., Certification letter_DHHS
letterhead_Salm_KH.doc). Add the current date and the name of the submitter to the cover letter. Change the
check mark as appropriate. Print out one copy to send to CDC.
5.10 Open the Certification Certificates.ppt file and personalize the appropriate certificate. Print out one copy to
send to CDC.
5.11 Record the information about the completed certification on the Excel sheet (e.g., “date certified,” “date
delivered to CDC”).
5.12 Add the completed certification to a running list of all certifications completed by the evaluator. Record the
name of the submitter and his/her laboratory, whether they were certified for gel and/or analysis, the
pathogen, the date of the report (date analyzed), the date delivered to CDC, and any comments about the
analysis or the TIFF.
5.13 Paperclip the cover letter, report, and certificate together. Send to the Database Team Leader (Kelley Hise) at
CDC. The Team Leader will distribute the certifications among the Database Team for review. Send an email
to the Database Team Leader (Kelley Hise). In the email, include a list of the certifications being sent to
CDC. List the pathogen, the certified individual’s name, the laboratory ID, and what the individual was
certified for (i.e., gels and analysis, gels only, or analysis only).
5.14 Paperclip all the analyses, emails, TIFFs, etc. together with the log-in and analysis sheet on top, and file in
evaluator’s files in the appropriate laboratory folder.
5.15 Review certification files.
5.15.1 When received by CDC, the Database Team member and Methods Validation Laboratory Team
member will review the reports in a timely manner in accordance with the certification instructions
sent out with the strains to the participating laboratories.
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5.15.2 The review will consist of checking the TIFF quality assessment and the comments associated with
each analysis discrepancy. The reviewer will only sign his/her name if he/she agrees with the
evaluator’s assessment of the certification files. If the reviewer does not agree with the evaluator’s
assessment, the reviewer must contact the evaluator and discuss modifying the certification report.
5.15.3 Once the Database Team reviewer approves a certification, he or she must send an email to the
PulseNet Technical Steward listing who was certified, their lab ID, organism for which they were
certified, and what they were certified for (gels, analysis, or both).
5.16 TIFF images that pass certification and review indicate that the submitter is gel certified. Bundle files that
pass certification and review indicate that the submitter is analysis certified. The submitter is considered
certified as long as the laboratory successfully completes annual proficiency testing. If the submitted
certification files do not pass the certification evaluation, the individual will need to review the
troubleshooting comments received from the evaluator and resubmit once results have improved. If the
submitter fails certification three times, the individual will not be allowed to submit again for six months.
Before resubmitting, the individual will be expected to work with CDC and/or their PulseNet Area
Laboratory until satisfactory results are achieved. This includes, but is not limited to troubleshooting and
training in the PulseNet PFGE protocols, BioNumerics and the PulseNet masterscripts.
6.
FLOW CHART:
7.
BIBLIOGRAPHY:
8.
CONTACTS:
8.1 Database Team Leader
Kelley Hise
KHise@cdc.gov
(404) 639-0704
8.2 PulseNet Technical Steward
Susan Hunter
SHunter@cdc.gov
(404) 639-1749
9.
AMENDMENTS:
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Appendix PNQ04-1.
Standard Comments for Certification and Proficiency Testing Reports
“General” Comments:
ƒ A gel image that fills the entire window on the imaging equipment screen (without cutting off wells or the bottom of the
gel) may improve analysis.
ƒ
A gel image that fills the entire window on the imaging equipment screen (without cutting off wells or the bottom of the
gel) may make analysis easier.
ƒ
Lanes X-Y are skewed slightly but the skew does not interfere with the analysis.
ƒ
The TIFF file was renamed <labID>051602.tif by evaluator for analysis. In the future, please name all files sent to
CDC according to the standardized PulseNet naming system. The first two digits should be the laboratory ID (e.g.,
GA), the second two digits should be the year (e.g., 04), and the last three digits should be the unique number of
the TIFF or bundle file submitted that year from your laboratory (e.g., 001).
Band Marking:
ƒ Consistency in band marking could be improved by marking thick bands as singlets unless two bands (a doublet) can be
visualized on the TIFF.
ƒ
Bands down to the last band of the standard (~20 kb) should be marked. If test bands are close to the last band of the
standard by a visual check of the TIFF, mark the band. Most labs are marking the bands at ~20 kb in lanes 7 and 8.
Sometimes these bands run a little above the standard and sometimes they run a little below the standard.
ƒ
Listeria 2003 (03-H8394): A singlet is marked at ~150 kb on bundle file. Some laboratories are resolving and marking 2
bands in this area. [The light band directly under the bright band at ~150 kb should be marked on the bundle file.]
[A singlet is marked at ~150 kb on bundle file. Some laboratories are resolving and marking the lighter area directly
underneath the dark band at ~150 kb as a second band.]
ƒ
Listeria 2003 (04-H8395): A doublet is marked at ~50 kb on bundle file. Some laboratories are resolving and marking a
third band at ~40 kb.
Standard Lanes:
ƒ Please include standard lanes in bundle file for certification.
ƒ
Please place standards in the assigned lanes described in the certification or proficiency testing protocols.
ƒ
The outer two standard lanes skewed inward, but the skew did not interfere with the analysis.
ƒ
Bands of some of the standard lanes (especially lanes 1 and 15) are lighter than bands of the test strains. This did not
affect analysis. However, more consistent standardization of cell concentrations among all test and standard strains
would improve gel quality.
ƒ
The band distortion in the first and last lanes could cause inaccurate analysis if the band placement was not adequately
checked during normalization.
ƒ
Although for normalization with standard strain H9812 the ninth band at ~180 kb is not marked, for band
finding, it should be marked as it appears on the TIFF. In this case, it should be marked as a single band.
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ƒ
E. coli 2003 H9812: The area at ~170 is marked as a single band on the bundle file, but appears as a doublet on the
TIFF.
ƒ
Listeria 2003: The top two bands in each standard lane are closer together than on other certification gels. Make sure
you are following the Listeria standardized protocol, especially the comb placement, for optimum band separation.
ƒ
E. coli 2003: In the H9812 standard lanes, a few laboratories are resolving doublets at ~170 kb, ~76 kb, and ~33
kb such as appears in lane 5 of this TIFF. Lane 5 has very good band resolution. When doublets are resolved in
the noted areas, make sure the area of the doublet marked during normalization is consistent with the area of
any single bands at the same position on other lanes. For areas resolved as doublets but normalized as one band,
you may wish to normalize using the midpoint between the two doublet bands. However, in band finding, the
bands should be marked as they appear on the TIFF.
Gel Not Clean/Shadow Bands Present:
ƒ Bands are difficult to see on the TIFF and mark on the bundle file because the gel background is not clean. Spots and
other debris may be reduced by cleaning all staining and destaining containers and preparing fresh reagents with clean
glassware. Make sure that all agarose goes into solution when it is heated with 0.5X TBE.
ƒ
Background from DNA degradation (i.e., smearing in the lanes) may be reduced by carefully preparing new plugs to
prevent shearing. Background may also occur because the agarose was too hot when the plugs were made or when the gel
was poured and/or because of poor quality reagents. Washing the plugs at least two more times with TE Buffer may also
reduce background.
ƒ
“Shadow” or “ghost” bands are present. These probably indicate incomplete restriction and should not be present on a
gel. If shadow bands appear on gels in the future, repeat restriction with more units of enzyme, for a longer amount of
time and/or with a different lot of enzyme. Wash the plugs at least two more times with TE Buffer before restriction is
repeated.
ƒ
The BlnI pattern of 24-98 contains an extra faint band at ~140 kb. This band has been present on certification TIFFs
received from other laboratories. This faint band may be the result of incomplete restriction or it could be due to a
mutation in the strain itself. If this certification set must be used again, repeat restriction with more units of enzyme, for a
longer amount of time and/or with a different lot of enzyme. Wash the plugs at least two more times with TE Buffer
before restriction is repeated. If that does not eliminate the faint band, you may wish to consider requesting a new 24-98
strain from CDC by emailing PFGE@cdc.gov.
Top Bands:
ƒ Salmonella 2003: The distance between the first and second bands of the patterns on the TIFF is less than on the
comparison TIFFs. This difference may make comparison of your gels to patterns in the national database difficult,
especially when the first bands of test strain patterns are near the top of the gel. For example, the distance difference
appears to have affected the pattern of CDC 61-99 (BlnI). On the TIFF, a thick band appears at ~1070 kb for CDC
61-99 (BlnI) where a doublet appears on the comparison TIFFs. Make sure you are running the gels according to the
standardized protocol on 1% SeaKem Gold agarose (BioWhittacker) with CHEF settings of 30 Kb – 700 Kb [2.2 s –
63.8 s] for Salmonella species. You may also try running the gel for a longer period of time (without cutting off
bottom bands). Contact Mary Ann Fair (mal3@cdc.gov) for help if this does not solve the problem.
Bottom Bands:
ƒ Running the gel so that the last band of the standard is approximately 1.0-1.5 cm from the bottom of the gel (per PulseNet
protocol) may improve separation of bands.
ƒ
The certification was marked unsatisfactory because the bottom bands of the standard are not visible on XX.tif and are
too low on XX.tif. Please run the gel so that the last band of the standard is approximately 1.0-1.5 cm from the bottom as
per the PulseNet standard PFGE protocol.
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The bottom bands of the standard are too low. The gel should be run so that the last band of the standard is approximately
1.0-1.5 cm from the bottom per the PulseNet standard PFGE protocol.
Resolution:
ƒ This is a good TIFF but could be improved by increasing band sharpness.
ƒ
Improvements in band resolution and gel background clarity will aid analysis and improve comparisons with the national
database.
ƒ
Fuzzy bands may occur because of an imaging problem or because the agarose was too hot when the plugs were made or
when the gel was poured, because of incomplete plug washing, and/or because of poor quality reagents. On future gels,
you may want to check your TIFF imaging by determining if a fluorescent ruler placed beside the gel is in focus. If the
ruler is not in focus, the bands on the gel may not be either. You may want to check the filter on your GelDoc system to
make sure it is clean.
ƒ
On this gel, it appears that the wells are out of focus. This may indicate that the bands are not focused. On future gels,
you may want to check your TIFF imaging by viewing the gel directly on the UV box or by determining if a fluorescent
ruler placed beside the gel is in focus. If the ruler is not in focus, the bands on the gel may not be either.
ƒ
Bands seem to disappear at the bottom of the gel. This affected the bundle file analysis.
ƒ
Lanes with bands missing at the bottom of the gel and/or where the pattern is light may require repeating restriction with
a larger plug slice, more units of enzyme, for a longer amount of time, and/or with a different lot of enzyme. Wash the
plugs at least two more times with TE Buffer to remove excess lysis reagents or other impurities. Preparing new plugs
with more concentrated cell suspensions may also improve band appearance. Light bands could also be the result of
prolonged exposure to UV light before capture of the image.
ƒ
The bands are distinct, but, in most lanes, very light. Better standardization of cell concentrations among all test and
standard strains would improve gel quality and TIFF imaging. Lanes where the pattern is light may require repeating
restriction with a larger plug slice or preparing new plugs with more concentrated cell suspensions. Light bands could
also be the result of prolonged exposure to UV light before capture of the image. [Can add: Make sure that the UV filter
is in place while photographing the gel and that fresh ethidium bromide solution in the proper concentration (stock of 10
mg/ml diluted to 1:10,000 or 10 microliters in 100 ml reagent grade/deionized water) is used for staining gels.]
ƒ
The bands tend to be very light at the bottom of the gel. Correcting this may require repeating restriction with a larger
plug slice, more units of enzyme, for a longer amount of time, and/or with a different lot of enzyme. You could also try
washing the plugs at least two more times with TE Buffer to remove excess lysis reagents or other impurities. Light bands
could also be the result of prolonged exposure to UV light before capture of the image.
ƒ
This gel appears to have a different band resolution than the comparison gels; some thick single bands on the comparison
patterns appear as doublets on this gel.
ƒ
Improving the resolution of the lower molecular weight bands would improve analysis.
ƒ
Bands are fused, fuzzy, and difficult to distinguish. Use of a comb with 10 mm-wide teeth (instead of 5.5 mm-wide teeth)
and running the gel for a longer time may improve the separation of bands.
ƒ
Better standardization of cell concentrations among all test and standard strains would improve gel quality.
ƒ
In some test lanes, thick bands (possibly due to a slight DNA overload) make doublets difficult to distinguish.
ƒ
There appears to be too much DNA in the lanes. Use smaller plug slices or prepare new plugs with less concentrated cell
suspensions to improve separation of bands.
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…to improve band appearance and separation for ease of analysis.
ƒ
The bands appear thick. This could be the result of too much DNA in the lanes or an over-integration issue in image
capture. Use smaller plug slices or prepare new plugs with less concentrated cell suspensions to improve separation of
bands. Compare your gel as it appears on the UV box to the TIFF image. If thick bands do not appear on the gel but do
appear on the TIFF image, refer to the GelDoc manual for ways to improve your TIFF imaging (e.g., saturated pixels
function).
ƒ
Listeria: Turning the comb around and running the gel so that the last band of the standard is approximately 1.0-1.5 cm
from the bottom of the gel may improve separation of bands.
ƒ
Several doublets and triplets are difficult to distinguish from single bands and from doublets, respectively. For Listeria
gels, the comb is turned around so that the teeth face the top of the gel; run the gel long enough so that the last band of the
standard is approximately 1.0-1.5 cm from the bottom of the gel to help improve resolution and separation of bands.
ƒ
Bands appear wavy. This could be due to one or more of the following: plug damage from the pipet tip or spatula during
removal from buffer or loading onto the gel; the comb is not clean; there are bubbles on the plug slices during loading on
a gel; the plugs are not firm or are too thin, both of which may cause increased likelihood of damage from a pipet tip.
Fragile plugs may be caused by the agarose concentration not being high enough or by overheated agarose.
ƒ
The bands appear wavy. Careful handling of the plugs during plug preparation and loading may reduce distortions. Make
sure that the plug slices are firmly attached to the comb before slowly pouring the agarose into the gel mold so as not to
dislodge the plug slices.
ƒ
Frowning bands could occur because of buffer flow or temperature fluctuations during the running of the gel, because the
gels sat too long before they were run, because the plugs were too thick, or for another unknown reason. Bands with more
curve than those on this TIFF would be difficult to analyze accurately.
ƒ
Several lanes contain bands that are distorted. Careful handling of the plugs during plug preparation and loading may
reduce distortions.
Analysis:
ƒ Comparing your band markings in the software to a hard copy of your original TIFF may improve analysis.
ƒ
Improvements in band resolution and gel background clarity will aid analysis and improve comparisons with the national
database.
ƒ
During the first step of analysis in BioNumerics (1. Strips), place the top of the green box frame directly under the wells
and the bottom of the green box frame at the bottom edge of the gel. This will standardize lane sizes, which may produce
more accurate normalizations and improve comparisons with the national database.
ƒ
Also during the first analysis step, using the linear adjustment under “Edit tone curve” may provide better band clarity for
analysis.
ƒ
The bands in the bundle appear dark compared to the bands in the comparison lanes. This may be the result of using the
“enhance weak band” feature in the “edit tone curve” option under Step 1 of the BioNumerics analysis. Too much weak
band enhancement may make analysis more difficult. Remember to compare your band markings in the software to a
hard copy of your original TIFF.
ƒ
The gel strips in XX.bdl are too wide. After the lanes are defined in BioNumerics, adjust the “thickness” under
“edit settings” to increase or decrease the thickness of the defined gel strips so that the left and right edges of the
strips are just inside the outer edge of the bands. This will ensure that the bands will appear in a size for proper
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analysis. If the gel strips are defined incorrectly (too wide), the resulting narrow bands with large white patches
on either side of the bands are difficult to analyze.
ƒ
Do not overmark bands during analysis. If you think an area may be a doublet but no white space is apparent in
the middle of the area or no indentations appear on the sides of the area separating two bands, mark the area as a
single band or rerun a gel that has better band resolution to check the area.
ƒ
To decide if an area is a doublet or single band, look on the TIFF for white space in the middle of the area or indentations
on the sides of the area separating two bands. If white space and/or indentations are present, mark the area as a doublet.
ƒ
Make sure the normalization markers are placed consistently from standard lane to standard lane at the bottom of
the TIFF where doublets are resolved (~30 kb). The normalization markers should be placed on the doublet in the
same way for each standard lane: either on the top band, the bottom band or in between the 2 bands of the
doublet.
ƒ
The band marking in the bundle was good but could be improved by the presence of sharper bands and more
consistency in band marking from lane to lane (e.g., marking thick bands as singlets unless two bands [a doublet]
can be visualized on the TIFF).
ƒ
One extra band is marked at >2000 kb in the following lanes: Lane 5 (H2446), Lane 4 (H8395), and Lane 9
(H8395). To prevent this from happening in the future, you could do one or both of the following:
a. When first going to Step 4 Bands in BioNumerics, look at the whole gel picture (without a zoom) and
delete obviously extraneous bands. Make sure you have taken off the normalization so that all extraneous
bands have been deleted. Note - It looks like you've drawn the top of your green box outlining the gel area
(in Step 1 BioNumerics) low. Try putting the top of the green box directly under the wells next time.
b. After band finding, run a dendrogram of all the analyzed lanes. Extraneous high bands (and low bands)
will show up easily on the dendrogram.
ƒ
In the bundle file, a doublet was marked at ~160 kb. This area is difficult to distinguish from a singlet on the TIFF – there
is little (if any) white area in the middle and no defined indentations on the side separating 2 bands. Most laboratories are
resolving and marking a singlet at this position.
ƒ
Compare the doublet marked at ~270 kb in lane 8 (CDC 24-98, BlnI) on the bundle file to the singlet marked at
~360 kb in lane 2 (CDC 16-98, XbaI). Because the bands in the two areas look similar on this TIFF, they should
be marked consistently. When determining whether a band is a singlet or a doublet, look for white space in
between the bands and/or indentations (i.e., shoulders) on the sides of the area separating two bands.
ƒ
Before linking a lane to the local database, make sure the fingerprint type (e.g., PFGE-XbaI, PFGE-BlnI, etc.) is
correct. Changing fingerprints is necessary when more than one enzyme is used for restriction on a particular gel.
If the lanes are linked with incorrect fingerprint types, possible duplicate entries in the database could occur. To
change the fingerprint type, right-click on the desired lane and select “Change fingerprint type of lane…” After
the fingerprint type is changed, you can link the lane to an entry in the database. Both fingerprint types should be
indicated with a green dot next to the one entry in the BioNumerics database.
ƒ
Previous TIFF files submitted on m/d/yy (AA.tif) and on m/d/yy (BB.tif) were unsatisfactory due to a large amount of
specks present on the TIFF (AA.tif) and due to curving, distorted bands (BB.tif). The previous bundle file submitted
(BB.bdl) contained too many bands at the bottom of CDC 24-98 and did not include the standard lanes.
ƒ
This report analyzes the bundle submitted mm/dd/yy. Previous bundles submitted mm/dd/yy and mm/dd/yy were
edited to delete extra bands. The auto band finding feature in BioNumerics marked many extra bands on this
TIFF. In addition, some single bands on the TIFF were marked as doublets in previous bundles.
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ƒ
This report analyzes the bundle submitted mm/dd/yy. A previous bundle submitted mm/dd/yy (AA.bdl) was
edited to mark bands at the bottom of the TIFF and to change a doublet to a single band, as it appeared on the
TIFF.
ƒ
AA.tif and AA.bdl were analyzed for this report. Previous TIFFs submitted mm/dd/yy (XX.tif) and mm/dd/yy
(YY.tif) were unsatisfactory due to poor band resolution and an incorrect pattern for 61-99 (BlnI), respectively.
ƒ
AA.bdl (submitted mm/dd/yy) was analyzed for this report. A previous bundle submitted mm/dd/yy (XX.bdl)
was unsatisfactory due to inaccurate band finding in the H9812 standard lanes. Lane 3 of AA.tif contains a repeat
of CDC 68-98.
ƒ
AA.bdl contained fields and experiments that are not in the standardized PulseNet scripts. After installing the
December 2003 Master Scripts, create PulseNet bundle files using the bundle file lightning bolt icon on the left
side of the BioNumerics screen (PulseNet BioNumerics Version 2.5 Training Manual, December 2003, Page
104). Bundle files created using this icon will contain only PulseNet fields.
ƒ
When you have edited your analysis, please create a PulseNet bundle (use the lightning bolt icon, see page 104 in
the Appendix of the Dec. 2003 CDC BioNumerics manual) and submit your new bundle (you may call it
XXb.bdl, if you wish) with your XX.tif file to PFGE@cdc.gov, including the words “E. coli certification” in the
subject line.
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1.
PURPOSE: To describe the procedure for the performance and evaluation of proficiency testing within
PulseNet. This is part of the PulseNet QA/QC program.
2.
SCOPE: This procedure applies to PulseNet personnel at participating laboratories who perform proficiency
testing and to contractors and CDC personnel who organize the proficiency testing rounds, ship proficiency
testing strains, evaluate and review the proficiency testing submissions, and send out proficiency testing
reports.
3.
DEFINITIONS/TERMS:
3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
3.10
3.11
3.12
3.13
3.14
3.15
3.16
3.17
4.
Proficiency Testing: An annual assessment of the quality of the work being performed in PulseNet
participating laboratories. For each pathogen, proficiency testing includes two parts – a “TIFF sent by CDC”
and a “TIFF generated by the participating laboratory (i.e., in-house TIFF).”
QA/QC: Quality Assurance/Quality Control
PFGE: Pulsed-field Gel Electrophoresis
TIFF: Tagged Image File Format. A file of a gel image that can be analyzed in BioNumerics.
Certification files: TIFF and/or bundle files submitted by PulseNet participating laboratories for certification
evaluation
SOP: Standard Operating Procedure
CDC: Centers for Disease Control and Prevention
Gel certified: Formerly “TIFF certified.” An individual or laboratory that is certified in laboratory methods
for PFGE and image acquisition
“TIFF generated by the participating laboratory” (also called the “in-house TIFF”): This is a part of the
proficiency testing program where laboratories run a gel and produce a TIFF of the gel that contains the
Salmonella Braenderup H9812 standards and the proficiency testing strain restricted with the primary and
secondary enzymes. The TIFF is analyzed and submitted to the on-line database. If no one in the laboratory
is analysis certified, please refer to section 4.2.1.2 for submission instructions.
Analysis certified: An individual who is certified in BioNumerics gel analysis
BioNumerics: Gel analysis software used by PulseNet
Proficiency Testing Survey and Round: An annual survey consists of two rounds of testing, a Fall round and
a Spring round
“TIFF sent by CDC”: This is a part of the proficiency testing program where all laboratories analyze the
same TIFF sent to them by CDC
Certification file evaluator: An individual who evaluates certification files
TIFF quality: The grading of the appearance and ease of analysis of a TIFF according to the PulseNet TIFF
Grading Guidelines. This is a main component of the evaluation of a TIFF submitted for certification
Proficiency testing packet: A package sent to the PulseNet participant with the results of his/her PT
evaluation. It includes a hard copy of the cover letter, report, in-house TIFF, and submission email
Certification file reviewer: An individual who reviews and signs off on the certification reports submitted by
the certification file evaluator
RESPONSIBILITIES
4.1 Individuals performing PulseNet-related work (i.e., preparing PFGE gels and/or analyzing TIFF images) must
submit certification file(s) and have them evaluated before being able to participate in the proficiency testing
program. See PNQ02 (SOP for Certification of PulseNet Personnel) for information on becoming certified.
4.2 Certified individuals at PulseNet participating laboratories must:
4.2.1
Perform proficiency testing annually for each pathogen in which they are certified, as part of continuing
in-house QA/QC. All certified individuals in a laboratory perform proficiency testing in-house, and one
set of results (from one individual) per pathogen per laboratory is chosen for upload/submission to the
on-line proficiency testing database at CDC.
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4.2.1.1 Gel certified individuals run and photograph a gel containing the proficiency testing strain for
each pathogen in which they are certified. The TIFF generated by the participating laboratory is
produced.
4.2.1.2 If the laboratory does not have an analysis certified individual on staff, the TIFF file generated
from the proficiency testing is sent via email to pfge@cdc.gov with the subject line of “PT
organism.” In addition, the lab must indicate what isolate is in what lane, including the
standards.
4.2.1.3 Analysis certified individuals analyze the TIFF generated by the participating laboratory and
the TIFF sent by CDC in BioNumerics for each pathogen in which they are certified. They
upload the analyses and TIFFs to the appropriate on-line proficiency testing database at CDC.
4.2.1.4 An individual certified in gels and analysis may produce the TIFF generated by the
participating laboratory, analyze the proficiency testing TIFFs, and upload the results.
4.2.2 Perform proficiency testing according to the current PulseNet proficiency testing protocol. This includes
correctly uploading results before the submission deadline.
4.3 Individuals organizing an upcoming proficiency testing round must:
4.3.1 Work with the CDC Database Team and Validation Laboratory personnel to create a list of certified
laboratories scheduled to participate, edit documentation for the proficiency testing round, gather the TIFFs
sent by CDC, select the proficiency testing strains, and contact the participating laboratories as stated under
the procedure section of this document.
4.4 Individuals shipping isolates for an upcoming proficiency testing round must:
4.4.1 Decide which strains will be sent out and their coding, find out if a TIFF sent by CDC must be run for a
particular pathogen, order and pick up shipment supplies, contact the CDC shipping department in advance
of the ship date, and prepare isolates for shipment as stated in the procedure section of this document.
4.5 Individuals evaluating proficiency testing results (evaluators) must:
4.5.1 Assess the TIFF quality and ease of analysis of the TIFFs submitted for proficiency testing and assess the
gel analysis, including band marking, of proficiency testing submissions. All TIFFs and analyses submitted
should be evaluated based on the current PulseNet standards of TIFF quality and gel analysis.
4.5.2 Evaluate proficiency testing submissions and submit a typed report, using the existing templates, to CDC
reviewers within the time frame written in the proficiency testing instructions sent to the participating
laboratories with the proficiency testing strains. In addition to a hard copy of the typed report, submit a
hard copy of the cover letter, TIFF generated by the participating laboratory, and submission email for each
pathogen from each laboratory performing proficiency testing.
4.5.3 Modify reports based on CDC reviewers’ comments, if necessary. Resubmit reports to CDC reviewers as
soon as possible.
4.5.4 Keep all reports in electronic files in an organized folder system and keep hard copies of the TIFFs sent by
CDC and any analyses (e.g., dendrograms, lane-to-lane comparisons) generated in files organized by
proficiency testing round.
4.6 Individuals reviewing proficiency testing packets submitted by the evaluator (reviewers) must:
4.6.1 Review the submitted packets within the time frame written in the proficiency testing instructions sent to
the participating laboratories with the proficiency testing strains.
4.6.2 Submit signed packets to the next reviewer or to the Database Team Coordinator.
4.7 Individuals sending out proficiency testing packets must:
4.7.1 Mail each organized packet to the participating laboratories and APHL according to the protocol for each
type of laboratory (see Appendix PNQ03-1).
4.7.2 Record the name of the individual performing the proficiency testing, the name of the individual to whom
the packet was mailed, and the date mailed on an electronic file. Send this file to the individuals
organizing, evaluating, and reviewing the proficiency testing reports as evidence that the reports were
mailed.
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CODE: PNQ04
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PROCEDURE
5.1 Individuals organizing an upcoming proficiency testing round:
5.1.1 Edit and send for CDC review the proficiency testing instructions, cover letters, and reports for use in the
upcoming round of testing.
5.1.2 Contact the Database Team members to supply the TIFFs sent by CDC. Validate the TIFFs sent by CDC.
5.1.3 Contact the CDC laboratorians in charge of the proficiency testing strains to select strains for the round.
5.1.4 Contact the individual shipping the proficiency testing strains and provide him/her with a list of
laboratories that will participate in the proficiency testing round. Also, provide electronic copies of the
finalized proficiency testing instructions, cover letter, and any additional mailings to go out with the
proficiency testing strains.
5.1.5 Contact the laboratories that are scheduled to participate in the upcoming round in advance of the shipment
of proficiency testing strains. Let them know when to expect the strains to arrive.
5.1.6 Contact the participating laboratories when the proficiency strains are shipped, including a notice of when
the proficiency testing results are due. Coordinate this email with the individual shipping the proficiency
testing strains.
5.1.7 Contact the participating laboratories two weeks before the proficiency testing results are due, giving
notice of the impending deadline.
5.2 Individuals shipping strains for proficiency testing:
5.2.1 At 4-6 weeks before strains are shipped:
5.2.1.1 Decide which strain (E. coli, Salmonella, Listeria, Shigella, and/or Campylobacter) will be sent to
certified labs for proficiency testing.
5.2.1.2 Reconstitute at least two lyophilized vials of each strain, pick 3 different colonies from each
isolation plate and make PFGE plugs from the six cultures.
5.2.1.3 Run a 10-well gel with three H9812 standards in lanes 1, 5, 10 and the 6 new PFGE plugs and
one previously tested PFGE plug of same strain according to standardized conditions for the
organism. This will confirm that the desired pattern is obtained and that it has not changed. If
pattern is correct, run a gel with H9812 standards in lanes 1 and 4 and PT strain restricted with
primary enzyme (XbaI, AscI) in lane 2 and secondary enzyme (BlnI, ApaI) in lane 3.
5.2.2 At 3-4 weeks before strains are shipped:
5.2.2.1 Coordinate with the Database Team and the individual organizing the proficiency testing round
about the “TIFFs sent by CDC.” If a suitable TIFF (excellent or good quality with H9812
standards in lanes 1 and 4 and an appropriate strain cut with the primary and secondary enzymes
in lanes 2 and 3, respectively) cannot be found by the Database Team, have the CDC laboratory
run a suitable TIFF of each organism that can be used for “the TIFF sent by CDC” for
BioNumerics analysis.
5.2.2.2 Send TIFF to the individual organizing the proficiency testing and the individual evaluating the
proficiency testing results to be sure that the gel image is satisfactory so there will be time to
repeat it or find another one, if necessary.
5.2.3 At 2-3 weeks before strains are shipped:
5.2.3.1 Get preliminary list of labs and which proficiency testing strains will be sent to each so an
estimate can be made of the number of cultures that will be sent. The list should be organized by
category of shipment. For example, E. coli only, Salmonella only, Listeria only, E. coli and
Salmonella, E. coli and Listeria, Salmonella and Listeria, and E. coli, Salmonella, and Listeria.
5.2.3.2 Decide on the coding of the proficiency testing strains:
5.2.3.2.1 Order about 20 more labels than needed for each strain by contacting the shipping
department with the appropriate information. Ask to proof the sample labels before they
are printed.
5.2.3.2.2 Replace the original label on the vials with the proficiency testing label (this can be done
the week before cultures are shipped).
5.2.3.2.3 Order and/or pick up enough of the following supplies for the shipments:
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5.2.3.2.3.1 Shipping form CDC57.7, Rev. 7/2004
5.2.3.2.3.2 Peel-off mailing labels, CDC 0.689 Rev. 12/96 (Check with someone in the
shipping department because they can supply labels with sender, mailstop and
phone number already printed.)
5.2.3.2.3.3 Styrofoam sleeves
5.2.3.2.3.4 8” x 8” zip lock bags
5.2.3.2.3.5 Large white business envelopes
5.2.3.3 Coordinate with the individual organizing the proficiency testing round to edit emails and
documents sent with proficiency testing cultures.
5.2.3.3.1 When the date for shipping and other pertinent information is confirmed, make copies of
all documents that will be sent with proficiency testing cultures for the number of
shipments plus two extra.
5.2.4 At 1-2 weeks before strains are shipped:
5.2.4.1 Contact Yvonne Stifel or someone else in shipping to determine if sending 20-25 packages on
two consecutive days (M, T or T, W) of the next week will be a problem because of lack of
shipping containers or personnel. Adjust shipment date if necessary.
5.2.4.2 Confirm and correct current email and delivery addresses and phone numbers of contact person at
each lab who will receive proficiency testing strains and/or TIFFs. This can be done in
coordination with the individual organizing the proficiency testing round.
5.2.5 On the Thursday or Friday of the week before the strains are shipped:
5.2.5.1 Separate lyophilized proficiency testing strains according to shipment type (1 culture, 2 cultures,
etc.) and put in labeled styrofoam sleeves (E and S; E, L and S, etc.). Refrigerate in wire baskets
or other containers according to category.
5.2.5.2 For domestic shipments, fill out information on Shipping Form (CDC57.7, Rev. 7/2004) and
address and phone number of recipient on mailing labels. Separate into appropriate categories.
5.2.5.2.1 If the same items are being shipped to 3 or more domestic labs, only one shipping form has
to be filled out per order; in the “ShipTo:” section, fill in the number of laboratories and
attach a separate sheet with a list of names, addresses and phone numbers of the labs
receiving the cultures. The individual mailing labels still have to be completed for each
lab.
5.2.5.2.2 International shipments require a separate shipping form for each laboratory and
“Declaration for Export of Biologicals, Chemicals, Equipment, or Technical Data” Form
(CDC 50.117 Rev. 3/2000) in triplicate. Include a copy of an Import Permit for each
country, if required. Spell out both the genus and species name (i.e., Escherichia coli, and
not E. coli) on the forms.
5.2.5.3 Insert cover letter and other appropriate paperwork in long white business envelope and write
name of individual from each lab who will receive the cultures on the outside of the envelope. Put
the envelope with cover letter and directions and the completed mailing label in separate zip lock
bags. Include “Declaration for Export…” form and Import Permit in zip-lock bag for international
shipment. Sort in groups, according to the cultures they will receive.
5.2.5.4 Set up database, log-ins and passwords for the proficiency testing strains and TIFFs. If the log-in
and password information will be included with the cultures, print information and include with
other paperwork for each lab.
5.2.6 On the day of shipment
5.2.6.1 Remove cultures from refrigerator and insert into appropriate zip-lock bag with label and
paperwork. Take to shipping department by 10:00 am so they will have time to pack and ship the
same day. Ask them to notify you if shipments will be delayed.
5.2.6.2 Coordinate with the individual organizing the proficiency testing to email laboratories on the day
cultures are shipped with the appropriate documents and TIFFs attached to the email. Request that
the labs let you know if the package does not arrive by Friday, so it can be tracked. The labs do
not send the shipping boxes back to CDC.
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5.3 Individuals at participating laboratories:
5.3.1 Must perform the proficiency testing according to the current proficiency testing instructions sent out with
the proficiency testing strains.
5.3.2 All certified individuals in a laboratory should perform proficiency testing annually as part of in-house
QA/QC. However, only one set of results per pathogen per laboratory should be chosen for
upload/submission to the on-line database at CDC.
5.3.3 Laboratories must submit proficiency testing TIFFs and analyses performed by certified individuals.
Results submitted by non-certified individuals will automatically fail the proficiency testing round.
5.3.4 All results must be submitted correctly by the submission deadline to avoid penalty points. Laboratories
that cannot meet the deadline may submit a request for extension to PFGE@cdc.gov before the submission
deadline. In any email correspondence to the PFGE inbox, type “proficiency testing” in the subject line.
5.3.5 Laboratories must submit a “submission email” to the PFGE inbox after their results are uploaded.
5.3.6 Laboratories must pass proficiency testing annually to maintain certification.
5.3.6.1 Laboratories that fail one proficiency testing round must submit the proficiency testing results for
that pathogen again using the same strains in the next round of testing. For example, laboratories
that fail in the Fall round must submit again in the Spring round.
5.3.6.2 Laboratories that fail two times in a row (e.g., Fall round and Spring round) will lose their
certification and must submit routine gels to PFGE@cdc.gov . Individuals at those laboratories
must submit new certification files and pass certification again before reinstatement of their
certification. Please see PNQ02 for PulseNet Certification information.
5.4 Individuals evaluating proficiency testing results (evaluators):
5.4.1 In the last week of the deadline for submissions, check the submissions. If a laboratory has not uploaded a
part of the proficiency testing, the evaluator should send an email notifying the laboratory of this error.
5.4.2 TIFF evaluation
5.4.2.1 After the deadline for submissions has passed, compile a list of all the TIFFs generated by the
participating laboratories and send to the Database Team Coordinator at CDC so that hard copies
can be made for the evaluator. The list can be quickly generated by copying screen shots of the
submissions in BioNumerics. The “Temp” field should contain the name of the TIFF generated
in-house by each laboratory.
5.4.2.2 TIFF grading is easier if all the TIFFs sent to the evaluator are printed the same way (e.g., have
black bands on a white background, fill the whole 8-1/2 by 11” paper).
5.4.2.3 Once the hard copies of the TIFFs are received by the evaluator, objectively grade the TIFFs
according to the PulseNet TIFF Grading Guidelines. TIFFs should receive a grade of excellent,
good, fair, or poor. Record comments for improvement on a removable note attached to the hard
copy of the TIFF. The TIFF grade and the comments will be transferred to the proficiency testing
report once all the information on the report is ready for transcription.
5.4.3 Gel analysis evaluation
5.4.3.1 Evaluate one pathogen at a time. Access the appropriate on-line database and call up all the
proficiency testing submissions for that round in BioNumerics. Open any bundles from
international laboratories that were received. Check to make sure all submissions are there, that
they are linked properly, and that the strain numbers are entered correctly into the “Key field.”
5.4.3.2 Divide all submissions for each pathogen into the two proficiency testing parts, the “TIFF sent by
CDC” and the “TIFF generated in-house.” Create a dendrogram for each enzyme of all the
submissions for each part of the proficiency testing.
5.4.3.3 Record any questions for the CDC Database Team as the evaluation proceeds. Email the list to
CDC after the initial evaluation is complete.
5.4.3.4 “TIFF sent by CDC” evaluation
5.4.3.4.1 Compare band markings to the CDC submission, which should be analyzed according to
the PulseNet Gel Analysis Guidelines.
5.4.3.4.2 On the dendrogram, circle any areas with discrepancies as compared to the CDC
submission. Record a note about the discrepancy and at what molecular weight the
discrepancy took place.
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5.4.3.4.3
Check for any normalization discrepancies. This could be indicated by bands on a
submission that are consistently lower or higher than other submissions received. For the
“TIFF sent by CDC,” all laboratories are analyzing the same TIFF, so all bands should be
the same down the dendrogram. Record any points to be deducted for normalization or if
the submission should be failed because of a normalization error.
5.4.3.4.4 At the bottom of the dendrogram, record any submissions that are to have band marking
points deducted, and record the number of deducted points.
5.4.3.4.4.1 Three points are deducted for each discrepancy ≥300 kb.
5.4.3.4.4.2 Two points are deducted for each discrepancy from 299-100 kb.
5.4.3.4.4.3 One point (up to four total) are deducted for each discrepancy <100 kb.
5.4.3.5 “TIFF generated in-house” evaluation
5.4.3.5.1 Look at the band markings compared to the CDC submission which should be analyzed
according to the PulseNet Gel Analysis Guidelines. This will help determine the quality of
the band resolution of the submissions.
5.4.3.5.2 Compare each submission (i.e., each lane) to the actual TIFF submitted by the
participating laboratory.
5.4.3.5.2.1 Note whether the band marking matches the TIFF, according to the PulseNet
Gel Analysis Guidelines. If the band marking does not match the TIFF, circle
the discrepancies and record a note on the dendrogram about the discrepancies
and at what molecular weight the discrepancies took place. If the band marking
matches the TIFF but is different from the majority of submissions received,
note that on the dendrogram as well.
5.4.3.5.2.2 Note whether the normalization is correct by looking at how the band marking
compares to the other submissions. Ask CDC to check the normalization of
any lanes that may contain normalization errors. Note normalization errors on
the dendrogram and the number of points (or a failure) that should be deducted
on the report.
5.4.3.5.3 At the bottom of the dendrogram, record any submissions that are to have points deducted
and record the number of deducted points. Band marking deductions are made only if the
band marking does not match the PulseNet Gel Analysis Guidelines.
5.4.3.5.3.1 Three points are deducted for each discrepancy ≥300 kb.
5.4.3.5.3.2 Two points are deducted for each discrepancy from 299-100 kb.
5.4.3.5.3.3 One point (up to four total) are deducted for each discrepancy <100 kb.
5.4.3.5.3.4 Up to five points total can be deducted per submission if the enzyme used is
incorrect (i.e., per lane).
5.4.4 Report writing
5.4.4.1 Create an Excel spreadsheet that shows the laboratories in the left column with the point totals for
each category of proficiency testing evaluation along the top.
5.4.4.1.1 Use the TIFF grading and the notes on the dendrograms to fill in the Excel sheet, making
Excel add up the total number of points for each laboratory.
5.4.4.1.2 Use the submission email sent by each laboratory to fill in the information on the Excel
sheet pertaining to the individual(s) who performed the proficiency testing and the
equipment used, etc.
5.4.4.2 Open the report template for a particular pathogen and “save as” the name of the template_LabID
(e.g., “PT report form_Fall 2004_CDC.xls”). The template should be set up to automatically add
up the number of points entered in the “No. of Points Received” column. Fill in one report for
each laboratory per pathogen.
5.4.4.2.1 Record the date the results were received and the laboratory ID.
5.4.4.2.2 Use the points tallied in the Excel spreadsheet created in 5.4.4.1 to fill in the “No. of
Points Received” column. Check that the number of points in the report matches the
number of points on the Excel spreadsheet created in 5.4.4.1.
5.4.4.2.3 Type in any comments written on the dendrogram in the “Comments” column next to the
appropriate section (e.g., normalization, band marking, etc.). The comments should
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5.4.6
5.4.7
5.4.8
5.4.9
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explain any point deductions and/or any PFGE patterns that did not match the majority of
submissions received.
5.4.4.2.4 Type in any TIFF grading comments (recorded on the removable note placed on the hard
copy of the TIFF in 5.4.2.3) that would explain the reason for the grade in the appropriate
section in the “Comments” column.
5.4.4.2.5 At the bottom of the report under “Comments”, type in any more suggestions for TIFF
improvement, any more detailed notes about the band marking or band resolution, or any
additional notes.
5.4.4.2.6 Type in the information at the bottom of the report (e.g., person who prepared gel,
equipment used, etc.).
5.4.4.2.7 Type in the date the report was written.
5.4.4.3 When all the reports are finished, print out each report, checking each one to make sure the page
break is in an appropriate spot. Sign each report.
Proficiency testing packet
5.4.5.1 Open the cover letter template for a particular pathogen. Change the date as necessary. Type in a
participant name and Laboratory ID and then print out the first page of the cover letter on CDC
letterhead. Do this for all participants submitting results for that particular pathogen.
5.4.5.2 Put together the hard copies of the proficiency testing packet for each laboratory and each
pathogen as follows: cover letter, report, in-house TIFF, submission email. Group them by
laboratory and paper clip together.
5.4.5.3 Send all packets to the CDC Database Team Coordinator for distribution within the timeline
stated in the proficiency testing instructions.
5.4.5.4 If any CDC reviewers wish to have the packet modified, do so as soon as possible and resubmit to
CDC.
Keep all reports in electronic files in a folder system organized by proficiency testing round.
Keep hard copies of the TIFFs sent by CDC and any analyses (e.g., dendrograms and lane-to-lane
comparisons) generated in files organized by proficiency testing round.
When all reports are finalized, create a summary document for CDC showing summary statistics for the
proficiency testing round (e.g., number of laboratories participating, number that failed, number of
excellent TIFFs, etc.).
Create summary documents for posting on the PulseNet Listserv. These documents should summarize the
equipment and enzyme manufacturers used for the proficiency testing survey and also show pertinent
information about the proficiency testing results.
5.5 Individuals reviewing proficiency testing packets (reviewers).
5.5.1 When received by CDC, a Database Team member and Methods Validation Laboratory Team member will
review the reports in a timely manner in accordance with the proficiency testing instructions sent out with
the strains to the participating laboratories. The last reviewer will submit the packets to the Database Team
Coordinator who will give them to the individual assigned to copy and mail the reports.
5.5.2 The review will consist of checking the TIFF quality assessment and the comments associated with each
analysis discrepancy. The reviewer will only sign his/her name if he/she agrees with the evaluator’s
assessment of the proficiency testing files. If the reviewer does not agree with the evaluator’s assessment,
the reviewer must contact the evaluator and discuss modifying the proficiency testing report.
5.6 Individual(s) sending out proficiency testing packets:
5.6.1 Make copies of each proficiency testing packet according to Appendix PNQ03-1.
5.6.2 In an electronic file, type in the name of the laboratory, individual to whom the packet was mailed, and the
date the packet was mailed. Email the file to the database team members and the individuals organizing,
evaluating, and reviewing the proficiency testing reports.
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6.
CODE: PNQ04
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09
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FLOW CHART:
Individual is certified in gels, analysis, or both gels and analysis.
Certification sets are available for E. coli O157, Salmonella,
Shigella, Listeria, and Campylobacter
• *In order to maintain certification, PT must be performed for each
organism in which an individual is certified
CDC mails PT strains to laboratories with at least one
certified person
•Fall round: E. coli O157, Salmonella, and Shigella
•Spring round: Campylobacter and Listeria
Each person performs PT*
• Laboratory chooses one set of
results to submit per organism
Run “In-house TIFF”*
Analyze “In house TIFF” and
upload results to appropriate PT
database*
Analyze “TIFF sent by CDC” and
upload results to appropriate PT
database*
Submissions evaluated, reports sent
to participants, and overall results
posted on PulseNet Listserv
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7.
BIBLIOGRAPHY:
8.
CONTACTS:
8.1 CDC PulseNet Database Team
(404) 639-4558
PFGE@cdc.gov
8.2 CDC Validation Laboratory Team
(404) 639-4558
PFGE@cdc.gov
8.3 Database Team Coordinator: Kelley Hise
(404) 639-0704
KHise@cdc.gov
8.4 Shipping: Cedric McCollins
(404) 639-0515
CMcCollins@cdc.gov
or
Yvonne Stifel
(404) 639-3355
YStivel@cdc.gov
9.
AMENDMENTS:
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Appendix PNQ03-1
Copying and Mailing Instructions for Completed Proficiency Testing Packets
For U.S. Public Health Labs:
One set of copies mailed to Shari Rolando. Shari will mail a copy to the lab directors.
CDC will file one copy and mail original to submitter.
USDA FSIS lab:
CDC will file one copy and mail original to submitter.
USDA ARS lab:
Original mailed to:
Dr. Charlene R. Jackson, Ph.D.
Microbiologist
Antimicrobial Resistance Research Unit
ARS, SAA, USDA
Russell Research Center
950 College Station Road
Athens, GA 30605
CDC will file one copy of report.
USDA AMS lab:
Original mailed to:
Dr. Michael D. Sussman, Ph.D., Molecular Biologist
USDA, AMS, S&T, National
Science Laboratory, Biotechnology Department
801 Summit Crossing Place, Suite B
Gastonia, NC 28054
CDC will file one copy of report.
FDA ORA or CFSAN lab:
Originals mailed to:
Atin Datta, Ph.D.
U.S. Food and Drug Administration
Office of Regulatory Affairs
Rm. 1241, HFC-140
5600 Fishers Lane
Rockville, MD 20857
Atin is responsible for sending to submitters.
Set of copies mailed to Christine Keys.
CDC will file one copy of each report.
FDA CVM lab:
Original mailed to Dr. Shaohua Zhao.
FDA
CVM/Division of Animal and Food Microbiology
8401 Muirkirk Rd.
Laurel, MD 20708
CDC will file one copy of report.
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PulseNet Canada:
Original mailed to Dr. Lai-King Ng.
Health Canada
National Microbiology Lab, Bacteriology/Enterics
1015 Arlington St.
Winnipeg, MB R3E 3R2
CANADA
CDC will file one copy of report.
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STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN
COULTER CEQ 8000 PLATFORM
CODE: PNQ05
Effective Date:
06
08
10
1. PURPOSE: To describe the procedure for certifying PulseNet personnel to enable full
participation in PulseNet MLVA activities.
2. SCOPE: This procedure applies to all PulseNet personnel performing MLVA and creating peak
files.
3. DEFINITIONS/TERMS:
MLVA: Multiple-locus variable-number tandem repeat analysis
DNA: Deoxyribonucleic acid
PCR: Polymerase chain reaction
CDC: Centers for Disease Control and Prevention
SOP: Standard Operating Procedure
4. RESPONSIBILITIES:
4.1 Individuals performing PulseNet MLVA-related work must submit certification file(s) and have
them reviewed before being able to submit peak files to the PulseNet MLVA Database.
4.1.1 Submitted certification files must document the submitter’s highest level of competence in
producing peak files.
4.1.2 Individuals can be certified for peak files only at this time
5. PROCEDURE:
5.1 PulseNet participants request the E. coli O157 and S. enterica serotypes Typhimurium and
Enteritidis MLVA certification sets from CDC (pfge@cdc.gov) if they do not already have them.
5.2 CDC sends the requested certification set and detailed instructions (see Appendices PNQ05-1
through PNQ05-5) to reconstitute the cultures and primers, make DNA templates, perform PCR
and fragment analysis and export the peak file from the sequencer in the “.CSV” format according
to the standardized laboratory protocol (PNL19, PNL21, and PNL27).
5.3 Peak file(s) are submitted to CDC for review. See Appendices PNQ05-2, PNQ05-3 and PNQ05-4
for submission instructions.
5.4 Submitters are notified in writing of the results of their certification file evaluation.
5.4.1 If the submitted certification file passes the certification evaluation, the submitter is considered
certified as long as they remain in their current laboratory and that laboratory successfully
completes annual proficiency testing. If a person relocates to a different PulseNet laboratory,
they must be recertified.
5.4.2 If the submitted certification files do not pass the certification evaluation
5.4.2.1 The individual will need to review the troubleshooting comments received from the
evaluator and resubmit once results have improved.
5.4.2.2 If the submitter fails certification three times, the individual will not be allowed to
submit again for six months. Before resubmitting, the individual will be expected to work
with CDC until satisfactory results are achieved. This includes, but is not limited to
troubleshooting and training in the PulseNet MLVA protocol.
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6. FLOW CHART:
7. BIBLIOGRAPHY:
8. CONTACTS:
8.1 Eija Hyytia-Trees, D.V.M., Ph.D.
CDC
PulseNet Methods Development and Reference Unit
(404) 639-3672
EHyytia-Trees@cdc.gov
8.2 Patti Lafon, M.S.
CDC
PulseNet Methods Development and Reference Unit
(404) 639-2828
PLafon@cdc.gov
9. AMENDMENTS:
9.1 Appendix PNQ05-3 Instructions: PulseNet Certification for MLVA Peak Files of S. Typhimurium Beckman Coulter CEQ 8000 Platform added 2/5/2008.
9.2 Appendix PNQ05-3: The internal ladder isolates were assigned CDC identification numbers
10/6/2009.
9.3 Appendix PNQ05-4: Primer reconstitution instructions were added 3/2/2010. A list of certification
package contents was added to the top of each PNQ05-2 and PNQ05-3.
9.4 Appendix PNQ05-4 was added 6/8/2010. This document details the certification procedure for the
S. enterica serotype Enteritidis MLVA. Former Appendix PNQ05-4 (primer reconstitution
instructions) was renamed PNQ05-5 and was amended to reflect the addition of S. Enteritidis
MLVA certification.
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Appendix PNQ05-1
Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) E. coli O157:H7 and S.
Typhimurium Cultures and S. enterica serotype Enteritidis cultures on TSA stabs
Biological Safety Warning: E. coli 0157:H7 and Salmonella strains are considered Level 2 biological
agents by the U.S. Department of Health and Human Services. Use appropriate precautions when
handling the vial or culture. Carry out laboratory work in a biological safety cabinet when applicable to
ensure aseptic conditions and personal safety.
Note: Store the lyophilized cultures and TSA stabs at 4°C in the dark until they are reconstituted and
subcultured.
Materials Needed:
Sterile sturdy forceps
1 ml pipetman
1 ml sterile pipet tips
1 µl sterile inoculating loop
Reagents Needed:
Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
Sterile grade reagent water or Trypticase Soy Broth (TSB)
70% isopropyl alcohol
Procedure for Reviving Cultures:
Lyophilized cultures: Day 1
1. Document the isolate number (s) and the date(s) lyophilized for your records. Wipe the aluminum
cover and outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically remove the
aluminum cover and rubber stopper from the vial containing the lyophilized culture. Wipe the outside
of the rubber stopper and neck of the vial with isopropyl alcohol before removing the stopper.
2. Re-suspend the lyophilized cells with 1.0 ml of sterile grade reagent water. Allow to stand for a few
minutes and/or mix gently to produce a uniform suspension. With an inoculating loop, streak a small
amount of this suspension onto a blood agar plate (BAP) and incubate at 37°C overnight.
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TSA stabs: Day 1
1. Document the isolate number (s) and the stabbing date for your records. Remove the cap and wipe
the neck of the vial with isopropyl alcohol.
2. With an inoculating loop, streak a small amount of growth onto a blood agar plate (BAP) and
incubate at 37°C overnight.
Days 2 and 3
1. Check the BAP; if the culture appears pure, pick an isolated colony, and streak it on a fresh BAP;
incubate at 37°C overnight. Use the growth from this plate to make DNA templates of the certification
strains. Transfer culture to fresh medium and incubate at 37°C overnight; this will ensure that the same
culture can be retested, if necessary.
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Appendix PNQ05-2
Instructions: PulseNet Certification for MLVA Peak Files of E. coli O157-Beckman Coulter CEQ
8000 Platform
Dear PulseNet Participant,
This package should contain:
16 vials of primers (please see Appendix PNQ05-5 for primer reconstitution instructions)
8 lyophilized cultures of E. coli O157:H7 (certification strains)
1 Positive control (EDL933)
2 Internal Ladder isolates (EC04PN0139 and EC04PN0570)
After the certification strains have been reconstituted according to the directions in Appendix PNQ051, streak each culture onto agar plates (overnight incubation at 37oC), pick an isolated colony, and
subculture to another plate. Use the growth from the second plate to make the DNA templates. Please
let me know if this package does not arrive in a satisfactory condition, or if the cultures are not viable.
Please, make a stock culture (freeze at -70°C) of each of the strains according to your laboratory’s
policy within 1 week from receiving them. Long-term storage of these cultures will ensure the
availability of the PulseNet certification set for future use, including MLVA certification of
additional personnel.
The strain numbers of the E. coli cultures are as follows:
CDC# 01-98 CDC# 05-98 CDC# 07-98 CDC# 08-98 CDC# 12-98 CDC# 24-98
CD# 11 (G5286) CDC# 48 (G7602)
Please follow these supplemental instructions for testing the certification isolates by MLVA.
Refer to the “Laboratory standard operating procedure for PulseNet MLVA of Shiga toxinproducing Escherichia coli O157 (STEC O157)-Beckman Coulter CEQ 8000 Platform (PNL19)”
for detailed instructions.
1. Make DNA templates from each test isolate, positive control EDL933, and internal ladder
isolates EC04PN0139 and EC04PN0570
2. Perform PCR and fragment analysis following the instructions of the standard protocol with
the possible exception of the primer concentration modifications your laboratory may have had
to make to optimize the PCR assays
a. Make sure to include your PulseNet laboratory ID (the unique identifier code that was
assigned to your laboratory by CDC PulseNet) in front of the CDC strain ID number
and your initials after the strain ID number, i.e. follow the strain ID format
labID_CDC01-98xx
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3. Export the peak file from the sequencer in the .CSV format. Please notice that in addition to
the certification set isolates, the peak file must also contain the positive control strain EDL933
in duplicate for each reaction, one negative control for each reaction, the internal ladder in
duplicate, and the molecular size standard peaks (D1 peaks).
a. Name the peak files according to the standardized PulseNet naming system:
Use the laboratory ID that was assigned to your laboratory by CDC PulseNet for the
first two to four letters of the file. The next 2 spaces will indicate the year the file was
generated. The next 4 spaces indicate the month and the date the run was performed.
For example: GA090219.csv is a peak file run on Feb 19th, 2009 at the GA Public
Health Laboratory. If multiple runs are performed on a same day, differentiate the
peak files using sequential numbers, for example GA090219-1, GA090219-2
4. Send the peak file to CDC PulseNet at pfge@cdc.gov within four weeks after receiving the
strains.
a. In the email to CDC, include E. coli O157 MLVA Certification in the subject line
Currently, for E. coli O157, an individual may be certified for peak file submission only. Once the E.
coli O157 national MLVA database is available on-line, individuals may also be certified for analysis.
After the peak files are submitted, the PulseNet certification file evaluator will analyze the files and
inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of
receiving the files. If the peak file is satisfactory, the person who submitted the file will be eligible to
send peak files to PulseNet for analysis. If the submitted certification files are not satisfactory, the
individual will need to review the troubleshooting comments received from the evaluator and resubmit
once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work
with CDC until satisfactory results are achieved.
Please let me know if you have questions or further clarification is needed.
Good luck,
Eija Trees, D.V.M., Ph.D.
Microbiologist
PulseNet Methods Development and Reference Unit
Enteric Diseases Laboratory Branch/CDC
Tel: 404-639-3672
E-mail: EHyytia-Trees@cdc.gov
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Appendix PNQ05-3
Instructions: PulseNet Certification for MLVA Peak Files of S. enterica serotype Typhimurium Beckman Coulter CEQ 8000 Platform
Dear PulseNet Participant,
This package should contain:
14 vials of primers (please see Appendix PNQ05-5 for primer reconstitution instructions)
8 lyophilized cultures of Salmonella Typhimurium (certification strains)
1 Positive control (LT2)
2 Internal Ladder isolates (2009K0825 and 2009K0826)
After the strains have been reconstituted according to the directions in Appendix PNQ05-1, streak each
culture onto agar plates (overnight incubation at 37oC), pick an isolated colony, and subculture to
another plate. Use the growth from the second plate to make the DNA templates. Please let me know if
this package does not arrive in a satisfactory condition, or if the cultures are not viable. Please, make a
stock culture (freeze at -70°C) of each of the strains according to your laboratory’s policy within 1
week from receiving them. Long-term storage of these cultures will ensure the availability of the
PulseNet certification set for future use, including MLVA certification of additional personnel.
The strain numbers of the S.enterica serotype Typhimurium cultures are as follows:
CDC# 61-99 CDC# 63-99 CDC# 76-99 CDC# 78-99 CDC# 80-99 CDC# 81-99
CD# 83-99 CDC# H8290
Please follow these supplemental instructions for testing the certification isolates by MLVA.
Refer to the “Laboratory standard operating procedure for PulseNet MLVA of Salmonella
enterica serotype Typhimurium (S. Typhimurium) - Beckman Coulter CEQ 8000 Platform
(PNL21)” for detailed instructions.
1. Make DNA templates from each test isolate, positive control LT2, and internal ladder
isolates 2009K0825 and 2009K0826
2. Perform PCR and fragment analysis following the instructions of the standard protocol with
the possible exception of the primer concentration modifications your laboratory may have had
to make to optimize the PCR assays
a. Make sure to include your PulseNet laboratory ID (the unique identifier code that was
assigned to your laboratory by CDC PulseNet) in front of the CDC strain ID number
and your initials after the strain ID number, i.e. follow the strain ID format
labID_CDC61-99xx
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3. Export the peak file from the sequencer in the .CSV format. Please notice that in addition to
the certification set isolates, the peak file must also contain the positive control strain LT2 in
duplicate for each reaction, one negative control for each reaction, the internal ladder in
duplicate, and the molecular size standard peaks (D1 peaks)
a. Name the peak files according to the standardized PulseNet naming system:
Use the laboratory ID that was assigned to your laboratory by CDC PulseNet for the
first two to four letters of the file. The next 2 spaces will indicate the year the file was
generated. The next 4 spaces indicate the month and the date the run was performed.
For example: GA090219.csv is a peak file run on Feb 19th, 2009 at the GA Public
Health Laboratory. If multiple runs are performed on a same day, differentiate the
peak files using sequential numbers, for example GA090219-1, GA090219-2
4. Send the peak file to CDC PulseNet at pfge@cdc.gov within four weeks after receiving the
strains.
a. In the email to CDC, include S. Typhimurium MLVA Certification in the subject
line
Currently, for S. Typhimurium, an individual may be certified for peak file submission only. Once the
S. Typhimurium national MLVA database is available on-line, individuals may also be certified for
analysis.
After the peak files are submitted, the PulseNet certification file evaluator will analyze the files and
inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of
receiving the files. If the peak file is satisfactory, the person who submitted the file will be eligible to
send peak files to PulseNet for analysis. If the submitted certification files are not satisfactory, the
individual will need to review the troubleshooting comments received from the evaluator and resubmit
once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work
with CDC until satisfactory results are achieved.
Please let me know if you have questions or further clarification is needed.
Good luck,
Eija Trees, D.V.M., Ph.D.
Microbiologist
PulseNet Methods Development and Reference Unit
Enteric Diseases Laboratory Branch/CDC
Tel: 404-639-3672
E-mail: EHyytia-Trees@cdc.gov
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AUTHORIZED BY:
Page 8 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN
COULTER CEQ 8000 PLATFORM
CODE: PNQ05
Effective Date:
06
08
10
Appendix PNQ05-4
Instructions: PulseNet Certification for MLVA Peak Files of S. enterica serotype Enteritidis Beckman Coulter CEQ 8000 Platform
Dear PulseNet Participant,
This package should contain:
14 vials of primers (please see Appendix PNQ05-5 for primer reconstitution instructions)
8 TSA stabs of Salmonella Enteritidis (certification strains)
1 Positive control (K1891)
2 Internal Ladder isolates (H9560 and 2010K0017)
According to the directions in Appendix PNQ06-1, streak each culture onto agar plates (overnight
incubation at 37oC), pick an isolated colony, and subculture to another plate. Use the growth from the
second plate to make the DNA templates. Please let me know if this package does not arrive in
satisfactory condition, or if the cultures are not viable. Please, make a stock culture (freeze at -70°C)
of each of the strains according to your laboratory’s policy within 1 week from receiving them.
Long-term storage of these cultures will ensure the availability of the PulseNet certification set
for future use, including MLVA certification of additional personnel.
The strain numbers of the S. enterica serotype Enteritidis cultures are as follows:
CDC# K2148
CDC# H9654
CDC# 2009K0432
CDC# K4417
CDC# K2158
CDC# K0746
CDC# K3307
CDC# J0932
Please follow these supplemental instructions for testing the certification isolates by MLVA.
Refer to the “Laboratory standard operating procedure for PulseNet MLVA of Salmonella
enterica serotype Enteritidis- Beckman Coulter CEQ 8000 Platform (PNL27)” for detailed
instructions.
1. Make DNA templates from each test isolate, positive control K1891, and internal ladder
isolates H9560 and 2010K0017.
2. Perform PCR and fragment analysis following the instructions of the standard protocol with
the possible exception of the primer concentration modifications your laboratory may have had
to make to optimize the PCR assays.
a. Make sure to include your PulseNet laboratory ID (the unique identifier code that was
assigned to your laboratory by CDC PulseNet) in front of the CDC strain ID number
and your initials after the strain ID number, i.e. follow the strain ID format
labID_CDCK2148xx.
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 9 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN
COULTER CEQ 8000 PLATFORM
CODE: PNQ05
Effective Date:
06
08
10
3. Export the peak file from the sequencer in the .CSV format. Please notice that in addition to
the certification set isolates, the peak file must also contain the positive control strain K1891 in
duplicate for each reaction, one negative control for each reaction, the internal ladder in
duplicate, and the molecular size standard peaks (D1 peaks).
a. Name the peak files according to the standardized PulseNet naming system:
Use the laboratory ID that was assigned to your laboratory by CDC PulseNet for
the first two to four letters of the file. The next 2 spaces will indicate the year the
file was generated. The next 4 spaces indicate the month and the date the run was
performed. For example: GA090219.txt is a peak file run on Feb 19th, 2009 at the
GA Public Health Laboratory. If you perform multiple runs on a same day,
differentiate the peak files using sequential numbers, for example GA090219-1,
GA090219-2.
4. Send the peak file to CDC PulseNet at pfge@cdc.gov within four weeks after receiving the
strains.
a. In the email to CDC, include S. Enteritidis MLVA Certification in the subject line.
Currently, for S. enterica serotype Enteritidis, an individual may be certified for peak file submission
only. Once the national S. Enteritidis MLVA database is available on-line, individuals may also be
certified for analysis.
After the peak files are submitted, the PulseNet certification file evaluator will analyze the files and
inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four weeks of
receiving the files. If the peak file is satisfactory, the person who submitted the file will be eligible to
send peak files to PulseNet for analysis. If the submitted certification files are not satisfactory, the
individual will need to review the troubleshooting comments received from the evaluator and resubmit
once results have improved. If the submitter fails certification three times, the individual will not be
allowed to submit again for six months. Before resubmitting, the individual will be expected to work
with CDC until satisfactory results are achieved.
Please let me know if you have questions or further clarification is needed.
Good luck,
Eija Trees, D.V.M., Ph.D.
Microbiologist
PulseNet Methods Development and Reference Unit
Enteric Diseases Laboratory Branch/CDC
Tel: 404-639-3672
E-mail: EHyytia-Trees@cdc.gov
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 10 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN
COULTER CEQ 8000 PLATFORM
CODE: PNQ05
Effective Date:
06
08
10
Appendix PNQ05-5
Instructions: Reconstitution of Primers for E. coli O157 and Salmonella enterica serotypes
Typhimurium and Enteritidis
E. coli O157:
Reconstitution of the primers:
The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock, please
reconstitute the primers by adding following amounts of distilled water:
Amount of primer in the vial
10 nM
40 nM
Water needed for a 100 µM stock
100 µl
400 µl
Preparation of the working concentrations from the 100 µM stock:
Working concentration:
25 µM
5 µM
2.5 µM
1 µM
Water (µl) + primer (µl)
30.0 + 10.0
47.5 + 2.5
48.75 + 1.25
99.0 + 1.0
Salmonella Typhimurium:
Reconstitution of the primers:
The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock, please
reconstitute the primers by adding following amounts of distilled water:
Amount of primer in the vial
10 nM
20 nM
40 nM
Water needed for a 100 µM stock
100 µl
200 µl
400 µl
Preparation of the working concentrations from the 100 µM stock:
Working concentration:
5 µM
2.5 µM
VERSION:
Water (µl) + primer (µl)
47.5 + 2.5
48.75 + 1.25
REPLACED BY:
AUTHORIZED BY:
Page 11 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE BECKMAN
COULTER CEQ 8000 PLATFORM
CODE: PNQ05
Effective Date:
06
08
10
Salmonella Enteritidis:
Reconstitution of the primers:
The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock, please
reconstitute the primers by adding following amounts of distilled water:
Amount of primer in the vial
10 nM
20 nM
Water needed for a 100 µM stock
100 µl
200 µl
Preparation of the working concentrations from the 100 µM stock:
Working concentration:
12.5 µM
2.5 µM
1 µM
VERSION:
Water (µl) + primer (µl)
43.75 + 6.25
48.75 + 1.25
99.0 + 1.0
REPLACED BY:
AUTHORIZED BY:
Page 12 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED
BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
CODE: PNQ06
Effective Date:
06
08
10
1. PURPOSE: To describe the procedure for certifying PulseNet personnel to enable full
participation in PulseNet MLVA activities.
2. SCOPE: This procedure applies to all PulseNet personnel performing MLVA and creating
peak files.
3. DEFINITIONS/TERMS:
3.1 MLVA: Multiple-locus variable-number tandem repeat analysis
3.2 PCR: Polymerase chain reaction
3.3 CDC: Centers for Disease Control and Prevention
3.4 SOP: Standard Operating Procedure
4. RESPONSIBILITIES:
4.1 Individuals performing PulseNet MLVA-related work must submit certification file(s) and
have them reviewed before being able to submit peak files to the PulseNet MLVA Database.
4.1.1 Submitted certification files must document the submitter’s highest level of competence
in producing peak files.
4.1.2 Individuals can be certified for peak files only at this time
5. PROCEDURE:
5.1 PulseNet participants request the E. coli O157 and Salmonella enterica serotypes
Typhimurium and Enteritidis MLVA certification sets from CDC (pfge@cdc.gov) if they do
not already have them.
5.2 CDC sends the requested certification set and detailed instructions (see Appendices PNQ06-1
through PNQ06-5) to reconstitute the cultures and primers, make DNA templates, perform
PCR and fragment analysis and export the peak file from the sequencer in the “.txt” format
according to the standardized laboratory protocols (PNL23, PNL24 and PNL26).
5.3 Peak file(s) are submitted to CDC for review. See Appendices PNQ06-2, PNQ06-3 and
PNQ06-4 for submission instructions.
5.4 Submitters are notified in writing of the results of their certification file evaluation.
5.4.1 If the submitted certification file passes the certification evaluation, the submitter is
considered certified as long as they remain in their current laboratory and that laboratory
successfully completes annual proficiency testing. If a person relocates to a different
PulseNet laboratory, they must be recertified.
5.4.2 If the submitted certification files do not pass the certification evaluation
5.4.2.1 The individual will need to review the troubleshooting comments received from
the evaluator and resubmit once results have improved.
5.4.2.2 If the submitter fails certification three times, the individual will not be allowed to
submit again for six months. Before resubmitting, the individual will be expected to
work with CDC until satisfactory results are achieved. This includes, but is not
limited to troubleshooting and training in the PulseNet MLVA protocol.
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 1 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED
BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
CODE: PNQ06
Effective Date:
06
08
10
6. FLOW CHART:
7. BIBLIOGRAPHY:
8. CONTACTS:
8.1 Eija Hyytia-Trees, D.V.M., Ph.D.
CDC
PulseNet Methods Development and Reference Unit
(404) 639-3672
EHyytia-Trees@cdc.gov
8.2 Patti Lafon, M.S.
CDC
PulseNet Methods Development and Reference Unit
(404) 639-2828
PLafon@cdc.gov
9. AMENDMENTS:
9.1 Appendix PNQ06-3 was added 10/1/2009. This document details the certification procedure
for the S. enterica serotype Typhimurium MLVA.
9.2 Appendix PNQ06-4: Primer reconstitution instructions were added 3/2/2010. A list of
certification package contents was added to the top of each PNQ06-2 and PNQ06-3.
9.3 Appendix PNQ06-4 was added 6/8/2010. This document details the certification procedure
for the S. enterica serotype Enteritidis MLVA. Former Appendix PNQ06-4 (primer
reconstitution instructions) was renamed PNQ06-5 and was amended to reflect the addition of S.
Enteritidis MLVA certification.
VERSION:
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Page 2 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED
BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
CODE: PNQ06
Effective Date:
06
08
10
Appendix PNQ06-1
Laboratory Protocol to Reconstitute Lyophilized (Freeze-Dried) E. coli O157:H7 and S.
enterica serotype Typhimurium Cultures and S. enterica serotype Enteritidis Cultures on
TSA Stabs
Biological Safety Warning: E. coli 0157:H7, S. enterica serotypes Typhimurium and Enteritidis
strains are considered Level 2 biological agents by the U.S. Department of Health and Human
Services. Use appropriate precautions when handling the vial or culture. Carry out laboratory
work in a biological safety cabinet when applicable to ensure aseptic conditions and personal
safety.
Note: Store the lyophilized cultures and TSA stabs at 4°C in the dark until they are reconstituted
and subcultured.
Materials Needed:
Sterile sturdy forceps
1 ml pipetman
1 ml sterile pipet tips
1 µl sterile inoculating loop
Reagents Needed:
Trypticase Soy + 5% Sheep Blood Agar plates (BAP) or equivalent media
Sterile grade reagent water or Trypticase Soy Broth (TSB)
70% isopropyl alcohol
Procedure for Reviving Cultures:
Lyophilized cultures: Day 1
1. Document the isolate number (s) and the date(s) lyophilized for your records. Wipe the
aluminum cover and outside of the vial with isopropyl alcohol. Using sturdy forceps, aseptically
remove the aluminum cover and rubber stopper from the vial containing the lyophilized culture.
Wipe the outside of the rubber stopper and neck of the vial with isopropyl alcohol before
removing the stopper.
2. Re-suspend the lyophilized cells with 1.0 ml of sterile grade reagent water. Allow to stand
for a few minutes and/or mix gently to produce a uniform suspension. With an inoculating loop,
streak a small amount of this suspension onto a blood agar plate (BAP) and incubate at 37°C
overnight.
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STANDARD OPERATING PROCEDURE FOR
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BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
CODE: PNQ06
Effective Date:
06
08
10
TSA stabs: Day 1
1. Document the isolate number (s) and the stabbing date for your records. Remove the cap and
wipe the neck of the vial with isopropyl alcohol.
2. With an inoculating loop, streak a small amount of growth onto a blood agar plate (BAP) and
incubate at 37°C overnight.
Days 2 and 3
1. Check the BAP; if the culture appears pure, pick an isolated colony, and streak it on a fresh
BAP; incubate at 37°C overnight. Use the growth from this plate to make DNA templates of the
certification strains. Transfer culture to fresh medium and incubate at 37°C overnight; this will
ensure that the same culture can be retested, if necessary.
VERSION:
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Page 4 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED
BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
CODE: PNQ06
Effective Date:
06
08
10
Appendix PNQ06-2
Instructions: PulseNet Certification for MLVA Peak Files of E. coli O157-Applied
Biosystems Genetic Analyzer 3130XL Platform
Dear PulseNet Participant,
This package should contain:
16 vials of primers (please see Appendix PNQ06-5 for primer reconstitution instructions)
8 lyophilized cultures of E. coli O157 (certification strains)
1 Positive control (EDL933)
2 Internal Ladder isolates (EC04PN0139 and EC04PN0570)
After the strains have been reconstituted according to the directions in Appendix PNQ06-1,
streak each culture onto agar plates (overnight incubation at 37oC), pick an isolated colony, and
subculture to another plate. Use the growth from the second plate to make the DNA templates.
Please let me know if this package does not arrive in satisfactory condition, or if the cultures are
not viable. Please, make a stock culture (freeze at -70°C) of each of the strains according to
your laboratory’s policy within 1 week from receiving them. Long-term storage of these
cultures will ensure the availability of the PulseNet certification set for future use,
including MLVA certification of additional personnel.
The strain numbers of the E. coli cultures are as follows:
CDC# 01-98 CDC# 05-98 CDC# 07-98 CDC# 08-98 CDC# 12-98 CDC# 24-98
CD# 11 (G5286) CDC# 48 (G7602)
Please follow these supplemental instructions for testing the certification isolates by
MLVA. Refer to the “Laboratory standard operating procedure for PulseNet MLVA of
Shiga toxin-producing Escherichia coli O157 (STEC O157)-Applied Biosystems Genetic
Analyzer 3130XL Platform (PNL23)” for detailed instructions.
1. Make DNA templates from each test isolate, positive control EDL933, and internal
ladder isolates EC04PN0139 and EC04PN0570.
2. Perform PCR and fragment analysis following the instructions of the standard protocol
with the possible exception of the primer concentration modifications your laboratory
may have had to make to optimize the PCR assays.
a. Make sure to include your PulseNet laboratory ID (the unique identifier code
that was assigned to your laboratory by CDC PulseNet) in front of the CDC strain
ID number and your initials after the strain ID number, i.e. follow the strain ID
format labID_CDC01-98xx.
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 5 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED
BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
CODE: PNQ06
Effective Date:
06
08
10
3. Export the peak file from the sequencer in the .txt (tab-delimited) format. Please notice
that in addition to the certification set isolates, the peak file must also contain the positive
control strain EDL933 in duplicate for each reaction, one negative control for each
reaction, the internal ladder in duplicate, and the molecular size standard peaks (R peaks).
a. Name the peak files according to the standardized PulseNet naming system:
Use the laboratory ID that was assigned to your laboratory by CDC
PulseNet for the first two to four letters of the file. The next 2 spaces will
indicate the year the file was generated. The next 4 spaces indicate the
month and the date the run was performed. For example: GA090219.txt is a
peak file run on Feb 19th, 2009 at the GA Public Health Laboratory. If you
perform multiple runs on a same day, differentiate the peak files using
sequential numbers, for example GA090219-1, GA090219-2
4. Send the peak file to CDC PulseNet at pfge@cdc.gov within four weeks after receiving
the strains.
a. In the email to CDC, include E. coli O157 MLVA Certification in the subject line.
Currently, for E. coli O157, an individual may be certified for peak file submission only. Once
the E. coli O157 national MLVA database is available on-line, individuals may also be certified
for analysis.
After the peak files are submitted, the PulseNet certification file evaluator will analyze the files
and inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four
weeks of receiving the files. If the peak file is satisfactory, the person who submitted the file will
be eligible to send peak files to PulseNet for analysis. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the
evaluator and resubmit once results have improved. If the submitter fails certification three
times, the individual will not be allowed to submit again for six months. Before resubmitting, the
individual will be expected to work with CDC until satisfactory results are achieved.
Please let me know if you have questions or further clarification is needed.
Good luck,
Eija Trees, D.V.M., Ph.D.
Microbiologist
PulseNet Methods Development and Reference Unit
Enteric Diseases Laboratory Branch/CDC
Tel: 404-639-3672
E-mail: EHyytia-Trees@cdc.gov
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 6 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED
BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
CODE: PNQ06
Effective Date:
06
08
10
Appendix PNQ06-3
Instructions: PulseNet Certification for MLVA Peak Files of S. enterica serotype
Typhimurium-Applied Biosystems Genetic Analyzer 3130XL Platform
Dear PulseNet Participant,
This package should contain:
14 vials of primers (please see Appendix PNQ06-5 for primer reconstitution instructions)
8 lyophilized cultures of Salmonella Typhimurium (certification strains)
1 Positive control (LT2)
2 Internal Ladder isolates (2009K0825 and 2009K0826)
After the strains have been reconstituted according to the directions in Appendix PNQ06-1,
streak each culture onto agar plates (overnight incubation at 37oC), pick an isolated colony, and
subculture to another plate. Use the growth from the second plate to make the DNA templates.
Please let me know if this package does not arrive in satisfactory condition, or if the cultures are
not viable. Please, make a stock culture (freeze at -70°C) of each of the strains according to
your laboratory’s policy within 1 week from receiving them. Long-term storage of these
cultures will ensure the availability of the PulseNet certification set for future use,
including MLVA certification of additional personnel.
The strain numbers of the S. enterica serotype Typhimurium cultures are as follows:
CDC# 61-99 CDC# 63-99 CDC# 76-99 CDC# 78-99 CDC# 80-99 CDC# 81-99
CDC# 83-99 CDC# H8290
Please follow these supplemental instructions for testing the certification isolates by
MLVA. Refer to the “Laboratory standard operating procedure for PulseNet MLVA of
Salmonella enterica serotype Typhimurium-Applied Biosystems Genetic Analyzer 3130XL
Platform (PNL24)” for detailed instructions.
1. Make DNA templates from each test isolate, positive control LT2, and internal ladder
isolates 2009K0825 and 2009K0826.
2. Perform PCR and fragment analysis following the instructions of the standard protocol
with the possible exception of the primer concentration modifications your laboratory
may have had to make to optimize the PCR assays.
a. Make sure to include your PulseNet laboratory ID (the unique identifier code
that was assigned to your laboratory by CDC PulseNet) in front of the CDC strain
ID number and your initials after the strain ID number, i.e. follow the strain ID
format labID_CDC61-99xx.
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 7 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED
BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
CODE: PNQ06
Effective Date:
06
08
10
3. Export the peak file from the sequencer in the .txt (tab-delimited) format. Please notice
that in addition to the certification set isolates, the peak file must also contain the positive
control strain LT2 in duplicate for each reaction, one negative control for each reaction,
the internal ladder in duplicate, and the molecular size standard peaks (R peaks).
a. Name the peak files according to the standardized PulseNet naming system:
Use the laboratory ID that was assigned to your laboratory by CDC
PulseNet for the first two to four letters of the file. The next 2 spaces will
indicate the year the file was generated. The next 4 spaces indicate the
month and the date the run was performed. For example: GA090219.txt is a
peak file run on Feb 19th, 2009 at the GA Public Health Laboratory. If you
perform multiple runs on a same day, differentiate the peak files using
sequential numbers, for example GA090219-1, GA090219-2.
4. Send the peak file to CDC PulseNet at pfge@cdc.gov within four weeks after receiving
the strains.
a. In the email to CDC, include S. Typhimurium MLVA Certification in the subject
line.
Currently, for S. enterica serotype Typhimurium, an individual may be certified for peak file
submission only. Once the national S. Typhimurium MLVA database is available on-line,
individuals may also be certified for analysis.
After the peak files are submitted, the PulseNet certification file evaluator will analyze the files
and inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four
weeks of receiving the files. If the peak file is satisfactory, the person who submitted the file will
be eligible to send peak files to PulseNet for analysis. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the
evaluator and resubmit once results have improved. If the submitter fails certification three
times, the individual will not be allowed to submit again for six months. Before resubmitting, the
individual will be expected to work with CDC until satisfactory results are achieved.
Please let me know if you have questions or further clarification is needed.
Good luck,
Eija Trees, D.V.M., Ph.D.
Microbiologist
PulseNet Methods Development and Reference Unit
Enteric Diseases Laboratory Branch/CDC
Tel: 404-639-3672
E-mail: EHyytia-Trees@cdc.gov
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 8 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED
BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
CODE: PNQ06
Effective Date:
06
08
10
Appendix PNQ06-4
Instructions: PulseNet Certification for MLVA Peak Files of S. enterica serotype
Enteritidis - Applied Biosystems Genetic Analyzer 3130XL Platform
Dear PulseNet Participant,
This package should contain:
14 vials of primers (please see Appendix PNQ06-5 for primer reconstitution instructions)
8 TSA stabs of Salmonella Enteritidis (certification strains)
1 Positive control (K1891)
2 Internal Ladder isolates (H9560 and 2010K0017)
According to the directions in Appendix PNQ06-1, streak each culture onto agar plates
(overnight incubation at 37oC), pick an isolated colony, and subculture to another plate. Use the
growth from the second plate to make the DNA templates. Please let me know if this package
does not arrive in satisfactory condition, or if the cultures are not viable. Please, make a stock
culture (freeze at -70°C) of each of the strains according to your laboratory’s policy within
1 week from receiving them. Long-term storage of these cultures will ensure the availability
of the PulseNet certification set for future use, including MLVA certification of additional
personnel.
The strain numbers of the S. enterica serotype Enteritidis cultures are as follows:
CDC# K2148
CDC# H9654
CDC# 2009K0432
CDC# K4417
CDC# K2158
CDC# K0746
CDC# K3307
CDC# J0932
Please follow these supplemental instructions for testing the certification isolates by
MLVA. Refer to the “Laboratory standard operating procedure for PulseNet MLVA of
Salmonella enterica serotype Enteritidis-Applied Biosystems Genetic Analyzer 3130XL
Platform (PNL26)” for detailed instructions.
1. Make DNA templates from each test isolate, positive control K1891, and internal
ladder isolates H9560 and 2010K0017.
2. Perform PCR and fragment analysis following the instructions of the standard protocol
with the possible exception of the primer concentration modifications your laboratory
may have had to make to optimize the PCR assays.
a. Make sure to include your PulseNet laboratory ID (the unique identifier code
that was assigned to your laboratory by CDC PulseNet) in front of the CDC strain
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 9 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED
BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
CODE: PNQ06
Effective Date:
06
08
10
ID number and your initials after the strain ID number, i.e. follow the strain ID
format labID_CDCK2148xx.
3. Export the peak file from the sequencer in the .txt (tab-delimited) format. Please notice
that in addition to the certification set isolates, the peak file must also contain the positive
control strain K1891 in duplicate for each reaction, one negative control for each
reaction, the internal ladder in duplicate, and the molecular size standard peaks (R peaks).
a. Name the peak files according to the standardized PulseNet naming system:
Use the laboratory ID that was assigned to your laboratory by CDC
PulseNet for the first two to four letters of the file. The next 2 spaces will
indicate the year the file was generated. The next 4 spaces indicate the
month and the date the run was performed. For example: GA090219.txt is a
peak file run on Feb 19th, 2009 at the GA Public Health Laboratory. If you
perform multiple runs on a same day, differentiate the peak files using
sequential numbers, for example GA090219-1, GA090219-2.
4. Send the peak file to CDC PulseNet at pfge@cdc.gov within four weeks after receiving
the strains.
a. In the email to CDC, include S. Enteritidis MLVA Certification in the subject line.
Currently, for S. enterica serotype Enteritidis, an individual may be certified for peak file
submission only. Once the national S. Enteritidis MLVA database is available on-line,
individuals may also be certified for analysis.
After the peak files are submitted, the PulseNet certification file evaluator will analyze the files
and inform your laboratory of your results (“Satisfactory” or “Needs Improvement”) within four
weeks of receiving the files. If the peak file is satisfactory, the person who submitted the file will
be eligible to send peak files to PulseNet for analysis. If the submitted certification files are not
satisfactory, the individual will need to review the troubleshooting comments received from the
evaluator and resubmit once results have improved. If the submitter fails certification three
times, the individual will not be allowed to submit again for six months. Before resubmitting, the
individual will be expected to work with CDC until satisfactory results are achieved.
Please let me know if you have questions or further clarification is needed.
Good luck,
Eija Trees, D.V.M., Ph.D.
Microbiologist
PulseNet Methods Development and Reference Unit
Enteric Diseases Laboratory Branch/CDC
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 10 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED
BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
CODE: PNQ06
Effective Date:
06
08
10
Tel: 404-639-3672
E-mail: EHyytia-Trees@cdc.gov
Appendix PNQ06-5
Instructions: Reconstitution of Primers for E. coli O157 and Salmonella enterica serotypes
Typhimurium and Enteritidis
E. coli O157:
Reconstitution of the primers:
The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock,
please reconstitute the primers by adding following amounts of distilled water:
Amount of primer in the vial
10 nM
40 nM
Water needed for a 100 µM stock
100 µl
400 µl
Preparation of the working concentrations from the 100 µM stock:
Working concentration:
25 µM
5 µM
2.5 µM
1 µM
Water (µl) + primer (µl)
30.0 + 10.0
47.5 + 2.5
48.75 + 1.25
99.0 + 1.0
Salmonella Typhimurium:
Reconstitution of the primers:
The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock,
please reconstitute the primers by adding following amounts of distilled water:
Amount of primer in the vial
10 nM
20 nM
40 nM
Water needed for a 100 µM stock
100 µl
200 µl
400 µl
Preparation of the working concentrations from the 100 µM stock:
VERSION:
REPLACED BY:
AUTHORIZED BY:
Page 11 of 12
STANDARD OPERATING PROCEDURE FOR
MLVA CERTIFICATION OF PULSENET PERSONNEL FOR THE APPLIED
BIOSYSTEMS GENETIC ANALYZER 3130XL PLATFORM
Working concentration:
25 µM
5 µM
2.5 µM
CODE: PNQ06
Effective Date:
06
08
10
Water (µl) + primer (µl)
30.0 + 10.0
47.5 + 2.5
48.75 + 1.25
Salmonella Enteritidis:
Reconstitution of the primers:
The amount of primer in each vial is indicated on the label. In order to prepare a 100 µM stock,
please reconstitute the primers by adding following amounts of distilled water:
Amount of primer in the vial
10 nM
20 nM
Water needed for a 100 µM stock
100 µl
200 µl
Preparation of the working concentrations from the 100 µM stock:
Working concentration:
12.5 µM
2.5 µM
VERSION:
Water (µl) + primer (µl)
43.75 + 6.25
48.75 + 1.25
REPLACED BY:
AUTHORIZED BY:
Page 12 of 12
READING CONTROL SHEET FOR:
STANDARD OPERATING PROCEDURES FOR THE PULSENET QA/QC
PROGRAM (PNQ)
NAME
DATE
COMMENTS
SIGNATURE
By signing above, you are indicating that you have read and understood all SOPs
included in the PNQ section of this manual.
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