User guide for the Escherichia coli combined Assay

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User guide for the
Escherichia coli combined Assay
Array Hybridisation Assay for DNA-based serogenotyping and detection of
resistance and virulence genes of Escherichia coli
For Research Use Only. Not Intended for Use in Clinical Diagnostics.
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CONTENT
BACKGROUND ................................................................................................................................. 1
Intended Use .............................................................................................................................. 2
Specifications .............................................................................................................................. 2
Technical Support ....................................................................................................................... 2
Safety Precautions ...................................................................................................................... 3
Material Safety Data Sheets (MSDS) .......................................................................................... 3
Shipping Precautions .................................................................................................................. 3
REAGENTS AND DEVICES ................................................................................................................. 4
Assay Components, Storage and Stability .................................................................................. 4
Cell Lysis (optional) ................................................................................................................... 4
DNA Labelling and Amplification .............................................................................................. 4
Hybridisation and Detection ..................................................................................................... 5
Instrumentation & Software .................................................................................................... 6
Components required but not provided .................................................................................. 6
PROTOCOL ....................................................................................................................................... 8
Culturing and Harvesting Bacterial Cells .................................................................................... 8
Extraction of DNA ....................................................................................................................... 8
Extraction of DNA by Spin Columns (e.g. Qiagen) .................................................................... 9
Linear Amplification and Internal Biotin Labelling ................................................................... 11
Hybridisation ............................................................................................................................ 12
General Remarks - Handling of Arrays ................................................................................... 12
General Remarks - Handling of Liquids .................................................................................. 12
General Remarks – the Substrate (Precipitating Dye) D1 ...................................................... 13
General Remarks - Thermoshakers ........................................................................................ 14
Protocol for Quantifoil’s BioShake iQ ..................................................................................... 14
Protocol for Eppendorf’s Thermomixer Comfort with microtitre plate adapter ................... 15
Data Analysis............................................................................................................................. 17
Starting the ArrayMate Reader .............................................................................................. 17
Worklist................................................................................................................................... 18
Data Acquisition in the ArrayMate Reader ............................................................................ 19
Results..................................................................................................................................... 21
Export of E. coli combined Test Reports ................................................................................. 22
TROUBLESHOOTING ...................................................................................................................... 24
Staining Control ........................................................................................................................ 24
Image Quality............................................................................................................................ 24
DNA Quality and RNA contamination control .......................................................................... 25
Physical Damage to the Array .................................................................................................. 25
Report Unavailable ................................................................................................................... 25
ADDITIONAL INFORMATION ......................................................................................................... 26
Warranty ................................................................................................................................... 26
Disclaimer ................................................................................................................................. 26
Quality Control ......................................................................................................................... 27
Legal Manufacturer .................................................................................................................. 27
Contact...................................................................................................................................... 27
E. coli combined Assay
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LITERATURE ................................................................................................................................... 28
UPDATES & SOFTWARE ................................................................................................................. 28
APPENDIX 1 – FLOW CHARTS ........................................................................................................ 29
APPENDIX 2 – PROBE TO TARGET TABLE ...................................................................................... 31
APPENDIX 3 – TYPING INFORMATION .......................................................................................... 41
Definitions & Explanations ....................................................................................................... 41
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BACKGROUND
The CLONDIAG Escherichia coli (E. coli) combined Assay allows DNA-based serogenotyping of
most known O- and H-antigens as well as, simultaneously, the detection of important antimicrobial resistance (i.e. blaOXA) and virulence genes (i.e stxA/stxB).
RNA-free, unfragmented genomic DNA from pure and monoclonal E. coli colony material is
amplified approximately 50-fold and internally labelled with biotin-11-dUTP using a linear
amplification protocol. In contrast to standard PCR, only one antisense primer per target is used
resulting in producing single stranded (ss) DNA reaction products. This allows simultaneous
sequence-specific labelling and amplification of an essentially unlimited number of targets.
However, sensitivity is lower than in a standard PCR (whereas contamination with amplicons is
nearly impossible) and for that reason the method is restricted to clonal colony material and
cannot be performed on samples such as swabs or other patient samples (e.g. faeces).
Resulting biotin labelled ssDNA is transferred and hybridised to DNA oligonucleotide
microarrays with 393 probes for different genetic markers and a biotin staining control. Probes
for serogenotyping are printed once, whereas probes for antimicrobial resistance (AMR) and
virulence genes are printed in two spots.
Targets include a variety of species and serotyping markers including genes that code for 23
different O-antigens and 47 H-antigens. Additionally, 102 probes targeting AMR genes as well
as 88 probes for virulence genes were included. Spot recognition is performed automatically
based on a digital image of the arrays and results are given as an html-file with a description of
each analysed target.
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GENERAL INSTRUCTIONS FOR USE
Intended Use
For Research Use Only. Not Intended for Use in Clinical Diagnostics.
This assay allows genotypic characterisation of E. coli isolates for research and epidemiological
applications. It must not be used as a substitute for phenotypic susceptibility tests and for the
guidance of antibiotic therapy.
Specifications
Upon receipt, the assay components need to be stored at different temperatures as specified
on the package insert. The assay is to be performed at an ambient temperature of 18°C to 28°C.
Technical Support
If you require any further information on this product please contact:
Alere Technologies GmbH
Löbstedter Straße 103 -105
D-07749 Jena - Germany
phone: +49 (0) 36 41 / 3111-0
fax: + 49 (0) 36 41 / 3111-120
cct.home@clondiag.com
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Safety Precautions
 The assay is intended for use by personnel that are trained in microbiological and
molecular methods. Preparation of DNA from pure E. coli colonies (clones) requires
expertise in microbiology and the local regulations for handling of pathogenic
microorganisms (biosafety level 2) are to be obeyed.
 Isolated, cell-free E. coli DNA may be processed without further biosafety precautions,
although contamination with E. coli or other bacteria needs to be ruled out.
 Always wear protective clothes as required for laboratory work by your local regulations.
Material Safety Data Sheets (MSDS)
According to OSHA 29CFR1910.1200, Commonwealth of Australia [NOHSC: 1005, 1008(1999)]
and the latest amendments to the European Union Directives 67/548/EC and 1999/45/EC, the
enclosed reagents do not require a Material Safety Data Sheet (MSDS). They do not contain
more than 1% of a component classified as hazardous and do not contain more than 0.1 % of a
component classified as carcinogenic. MSDS therefore are not provided. Nevertheless, the
buffers may cause irritation if they come into contact with eyes or skin, and may cause harm if
swallowed. The regular precautions associated with laboratory work should be obeyed (e.g.,
wear protective goggles, gloves and lab coat and avoid contact with the reagents). In case, any
liquids are spilled, clean with disinfectant and/or laboratory detergent and water.
Alere assumes no liability for damage resulting from handling or contact with these products. If
you have any questions please contact our Technical Support (see above).
Shipping Precautions
RID/ADR: Kein Gefahrgut / No dangerous goods
IMDG: No dangerous goods
IATA: No dangerous goods
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REAGENTS AND DEVICES
Assay Components, Storage and Stability
All reagents are provided in a certain surplus amount (see below). In case of need, all
components may also be ordered separately; please refer to the order numbers at the end of
this user guide. For pricing please contact your local representative or our customer service,
respectively.
The expiry date can be found on each bottle and on the outer package. All components have
been tested for stability for short term shipment (<1 week) at ambient temperature (< 37 °C).
The assay components with a rather limited stability are D1 and C3. The other components
have proven to be stable even six months after the assay expiry date has passed.
Cell Lysis (optional)

A1: Lysis Buffer (cat# 245101000)
Store at 18-28°C (ambient temperature). Surplus: 50%.

A2: Lysis Enhancer (lyophilised, cat# 245102000)
Store at 18-28°C (ambient temperature). Centrifuge A2 tubes shortly prior to opening. Add
200 µL Buffer A1 to Lysis Enhancer before use. Mix well and store for less than 1 week at 28°C. Sufficient for 96 isolations.
DNA Labelling and Amplification

B1+: Labelling Buffer, Store at 2-8°C. Surplus: 25%.

B2: Labelling Enzyme, Store at 2-8°C. Surplus: 50%.

Ecoli-PM1: lyophilized Labelling-Primer-Mix,
dilute in 50 µl molecular grade water, Store at -20°C,
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Hybridisation and Detection

ArrayStrips (12 x 8 samples),
Protected against light and sealed under inert gas. Store at 18°C to 28°C. After opening to
be used within two weeks. Close the unused wells with caps, protect them against
humidity and dust and store them at a dark place. Avoid any touching or scratching of the
surface of the microarray at the bottom of the well. Do not store or handle unused wells at
an air humidity of more than 60% since this may irreversibly corrode the spots.

CapStrips (24 strips)

C1: Hybridisation Buffer
Store at 18-28 °C, protect against sunlight. Surplus: 100%.

C2: Washing Buffer 1
Store at 18 °C - 28 °C, protect against direct sunlight. Surplus: 100%.

C3: HRP Conjugate 100x
Store at 2-8 °C, protect against direct sunlight. Surplus: 100%.

C4: Conjugate Buffer
Store at 18°C to 28°C, protect against direct sunlight. Surplus: 200%.

C5: Washing Buffer 2
Store at 18°C to 28°C, protect against direct sunlight. Surplus: 200%.

D1: Horseradish Peroxidase Substrate
Store at 2-8°C, protect against direct sunlight. Surplus: 50%.

CM: Reference DNA from E. coli EDL933 (GenBank accession number NC_002655.2),
cDNA = 0.1-0.4 µg/µL. Store at 2-8 °C. Sufficient for 5-6 tests.
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Instrumentation & Software

ArrayMate
Reader
(to
be
ordered
separately,
for
details
see
below)
The E. coli combined assay may be used on the ArrayMate reader only. The alternative
devices ATR01/03 are not suitable for reading ArrayStrip based assays. In case of any
questions please contact us.

Iconoclust software (provided with the reader)

Test specific software plug-in (can be downloaded from Alere Technologies’/CLONDIAG´s
website, check periodically for updates, for details see below). Information (such as spot
names, marker names, location of the spots on the array, size of the image taken by the
reader’s specific camera) is delivered with the reader or can be downloaded from our
website. These test-specific plug-ins will occasionally be updated. Please check the NEWS
section of our website http://www.alere-technologies.com. Support is available via
cct.home@clondiag.com.
Components required but not provided

Growth media for the cultivation of E. coli. The test should be performed with colonies
harvested from 2xTY or Columbia Blood Agar. Other rich media (e.g. Standard 1 or LB) may
also suffice, but have not systematically been tested. Liquid media should not been used
because contaminations or mixed cultures cannot easily be ruled out.

Equipment and consumables needed for the cultivation of E. coli (incubator, inoculation
loops, Petri dishes)

DNA preparation kits:
The assay has been tested with the DNeasy Blood & Tissue Kit from Qiagen (cat# 69504)
and High Pure DNA Isolations Kit from Roche (Cat. No. 11796828001).
Please note:
The DNA specimen needs to be free of RNA. Recommendation: a pretreatment with the Cell Lysis components A1/A2 (see below) or a standard
RNase A treatment while DNA preparation.
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
Equipment needed for DNA isolation, e.g. pipettes, centrifuge, thermoshaker or automated
device (see above)

Photometer (OD 260 nm) for measuring the concentration of DNA

Equipment for non denaturing agarose DNA gel electrophoresis for quality control of DNA

Thermocycler for PCR

Thermoshaker
We
strongly
recommend
the
BioShake
iQ
by
Quantifoil
Instruments
(http://www.qinstruments.com/) equipped with a customised heating block designed to fit
ArrayStrips. Alternatively, you may use Eppendorf’s Thermomixer Comfort, equipped a
heating block for microtitre plates.

Pipettes: suitable for 1µL-5µL volumes, 90µL, 100µL, 200µL, 1000µL

Multichannel Pipettes for 100-200 µL

Reaction vials suitable for PCR

Ultrapure (PCR grade) water

RNase A (we recommend Qiagen’s RNase A solution, 100 mg/mL, Qiagen order# 19101).

Pasteur pipettes (BRANDT / Cat. No. 612-2856).
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PROTOCOL
Culturing and Harvesting Bacterial Cells
Members of the genus Escherichia are potential pathogens. All procedures for cultivation of
the bacterium and DNA preparation need to be performed by properly trained staff in a
biosafety level 2 facility.
Grow E. coli on 2xTY or Columbia Blood agar (overnight at 37°C or 48 hrs at room temperature).
Obtain confirmation of the identification as E. coli (API, VITEK, MALDI) and make sure that you
have a pure, monoclonal culture of E. coli. Contamination with other bacteria, especially with
other Enterobacteriaceae needs to be strictly avoided as the might carry the same resistance
genes as certain E. coli strains and thus can introduce false positive signals and patterns.
Extraction of DNA
The required sample type for the E. coli combined assay is 0.5-2 µg (cDNA = 0.1-0.4 µg/µl) of
intact genomic DNA from a single clone.
The DNA specimen needs to be free of RNA and it should not be fragmented. This can be
determined by agarose gel electrophoresis. Additionally, the microarray includes probes as an
internal control for RNA contamination. The automatic software analysis will give a “failed” if a
RNA contamination occurred while DNA isolation.
DNA should not be prepared by disrupting E. coli cells using bead beaters, ultrasonication or
aggressive chemicals such as in alkaline lysis protocols. Most performance problems with the
E. coli combined assay are due to insufficient amounts or quality of DNA preparation. We
therefore strongly recommend following the protocols outlined below.
Please note: To yield more genomic DNA, we recommend the optional cell lysis step with
A1/A2 reagent.
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Extraction of DNA by Spin Columns (e.g. Qiagen)

Add an inoculating loop full of monoclonal colony material of the E. coli isolate to 0.2 ml
1xPBS and vortex thoroughly.
Loop empty
Loop full
It is important to harvest enough bacteria; this is a prerequisite
for extraction of a sufficient amount of DNA.
Take an inoculating loop of 1mm diameter filled with bacteria as
shown in the right picture.
Optional cell lysis with A1/A2 reagent (instead of 1xPBS):
 Centrifuge A2 tube shortly, open it, add 0.2 ml of Lysis Buffer A1 to Lysis Enhancer A2
and dissolve.
 Add an inoculating loop full of monoclonal colony material of the E. coli isolate to this
A1/A2 reagent and vortex thoroughly.
 Incubate the colony material of the E. coli isolate in A1/A2 for 30-60 min at 37°C and 550
rpm in the thermoshaker.

Proceed with the DNA preparation protocol of the DNA preparation kit. For the Qiagen
DNeasy Blood&Tissue Kit that is as follows:

Add 20 µL proteinase K (Qiagen Kit, or equivalent) and add 200 µL buffer AL (Qiagen Kit)

Vortex shortly or shake vigorously.

Incubate for 30-60 min at 56°C and 550 rpm in the thermoshaker.

important: if A1/A2 reagent not used, add now 4 μl RNase A (100 mg/ml), mix by
vortexing, and incubate for 2 min at room temperature before continuing

Add 200 µl ethanol (96-100%)

Vortex the sample and centrifuge shortly.
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
Transfer the complete content of the tube (including any precipitate) into a spin column
that is placed in a 2 ml collection tube.

Centrifuge (8000 rpm, 1 min) at room temperature, time and speed need to be determined
depending on viscosity of the sample and type of centrifuge used. All liquid should be
collected in the collection tube afterwards.

Discard collection tube with liquids.

Place the spin column in a new 2 ml collection tube (provided with the kit).

Add 500 µl Buffer AW1.

Centrifuge (8.000 rpm, 1 min) at room temperature.

Discard collection tube with liquids.

Place the spin column in a new 2 ml collection tube (provided with the kit).

Add 500 µl Buffer AW2.

Centrifuge (14.000 rpm, 3 min) at room temperature, the membrane of the spin column
should be dry, and all liquid should be in the collection tube.

Discard collection tube with liquids.

Place the spin column in a clean 1.5 ml tube (not provided with the kit).

Add 100 µl Buffer AE (or PCR grade distilled water) directly onto the membrane of the spin
column.

Incubate at room temperature for 1 min to elute DNA.

Centrifuge (8000 rpm, 1min) at room temperature.

Optional: add another 100 µl Buffer AE (or PCR grade distilled water) directly onto the
membrane, incubate at room temperature for 1 min and centrifuge again.

Discard the spin column.
Please note: Ethanol from Washing Buffers strongly inhibits the enzymes used in the assay.
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A contamination with Washing Buffer might occur during elution of prepared DNA by drops
adhering to the funnel of the spin columns. Thus these funnels should be gently touched and
tried with sterile filter paper or wipes prior to the elution step. Alternatively, prepared DNA can
shortly be heated to evaporate ethanol (e.g., 10 min at 70 C).

Check for DNA integrity and absence of RNA (e.g., agarose gel). If necessary, you might
perform another digestion step with additional RNase A (not provided). Measure DNA
concentration (A260 method), it shouldn´t be less than 0.1 µg/µl. The concentration might
be increased by heating and evaporation of water, or by using a speed vac centrifuge.
Linear Amplification and Internal Biotin Labelling
Please keep in mind the limited surplus of reagents whilst pipetting. The surplus of B1+ labelling
reagent is 25 %.

Prepare a Master Mix by combining 3.9 µL of B1+ labelling reagent, 1 µl Ecoli-PM1
Labelling-Primer-Mix and 0.1 µL of B2 (DNA polymerase) per sample.

Add 5 µl of E. coli DNA (cDNA = 0.1 - 0.4 µg/µl) prepared as described above to 5 µl of the
Master Mix (B1+/B2). Do not forget to label the vial!

Perform amplification in a pre-programmed thermocycler (e.g., Eppendorf Mastercycler
gradient with heated lid, VWR, cat No. 460-0108) according to following protocol:
Pre-heat cover/lid to 105°C
300 sec at 96°C
20 sec at 50°C
50 cycles with
40 sec at 72°C
60 sec at 96°C
Cool down to 4°C, hold

the samples can be stored frozen until usage
Please note: When using another device, some adaptations might be necessary. Before
starting routine use, please test the protocol with a few known reference strains.
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Hybridisation
General Remarks - Handling of Arrays

Never touch the array surface!

Avoid complete drying of the array surface during processing!

Do not allow it to stay without liquid for more than two minutes!

Never rinse the wells with distilled water after the hybridisation step, use only C2
Washing Buffer!
Unused wells should be capped during the whole procedure. The strips may be processed up to
three times without a loss of quality of properly capped unused arrays. Close all wells that will
not be used with a cap und leave it there until you use these wells (for storage conditions after
use: see section “Assay components, storage and stability/Hybridisation and Detection”).
Always label your array strips with a laboratory marker at the recommended position. Never
label them on the bottom or across the data matrix barcode! This may cause an error.
Barcode
label HERE
(keep it clean!)
Avoid contact of data matrix barcode with organic solvents! The ArrayMate needs the
information encoded in the data matrix to perform the assay and the analysis afterwards.
Avoid touching the bottom of the microarray strip and keep it clean.
General Remarks - Handling of Liquids
We recommend the use of a multichannel pipette and reagent reservoirs. Please keep in mind
the limited surplus of C1 (100%).
We strongly recommend that the liquid is removed by pipetting rather than by inverting the
strips and flicking the liquids out. Fine tipped soft, disposable Pasteur pipettes are suited best
(such as BRANDT / Cat. No. 612-2856). Always place the pipette tip at the cavity between the
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array and the wall of the reagent well. If you touch the array surface, probes may be scratched
off and this may cause an error.
Pipette
tip
use the cavity between array
and the wall of the tube
do never touch the array
Array
General Remarks – the Substrate (Precipitating Dye) D1
An appropriate amount of substrate (precipitating dye) should be filled into an Eppendorf tube
and taken out of the refrigerator when starting the procedure allowing it to pre-warm to room
temperature/25°C. Cold D1 may yield weak signals. D1 should shortly be centrifuged prior to
use to remove bubbles as well as possible precipitates.
Triggered by peroxidase, in case of positive reactions, the dye precipitates but it is not
covalently bound. The precipitate can be dissolved by vigorous shaking. Thus the arrays must
not be shaken, dropped or moved abruptly during the staining procedure and afterwards.
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After completion of staining, remove and discard reagent D1 as completely as possible and scan
immediately. The dye precipitate fades slowly in presence of liquids.
General Remarks - Thermoshakers
The correct temperature within the vessels is essential; therefore always use the appropriate
equipment for heating. Because of a possibly inhomogeneous distribution of the temperature
within the heating block and because of possible differences between displayed and actual
temperatures, the use of different brands of thermoshakers might affect test performance. We
tested the assay with BioShake iQ by Quantifoil Instruments (http://www.qinstruments.com/)
equipped with a customised heating block designed to fit ArrayStrips and Eppendorf’s
Thermomixer Comfort, equipped with a heating block for microtitre plates.When using other
devices, some modifications to the protocol might be necessary. Before starting routine use,
please test the protocol with a few known reference strains or the control DNA (E. coli EDL933).
Protocol for Quantifoil’s BioShake iQ

Switch on the thermoshaker and let it pre-heat to 50°C.

Remove the ArrayStrip(s) from the pouch.

Insert the ArrayStrip(s) into the white frame. Assure the correct orientation (data matrix
barcode close to row (A) and proper fit.

Pre-wash the array in two steps:

First, PCR-grade distilled water, 200 µl per well at 50°C, 5 min and 550 rpm

Second, C1 Hybridisation Buffer, 150 µl per well at 50°C, 5 min and 550 rpm

Add 90 µL of buffer C1 to each tube with (10 µL) labelled amplification product, mix gently

Remove the buffer from the well with the array and add the mixture of C1 and labelled
amplification product

Incubate at 50°C, 60 min and 550 rpm.
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
Remove liquid and add 200 µl C2 Washing Buffer. Incubate at 45°C, 10 min and 550 rpm,
remove and discard.

Add another 200 µl C2 Washing Buffer. Incubate at 45°C, 10 min and 550 rpm, remove and
discard.

Meanwhile, prepare conjugate: for each experiment add 1 µl conjugate 100xHRP to 100 µl
C4 Conjugation Buffer. This mixture is stable for around one working day at room
temperature; C3 is delivered with a surplus of 100%, C4 is delivered with a surplus of 200%.
Suggested pipetting scheme:
C3
C4

1
well
1.5 µL
150 µL
2-3
wells
3.5 µL
350 µL
4-6
wells
7 µL
700 µL
7-10
wells
11 µL
1100 µL
11-15
wells
16 µL
1600 µL
16-20
wells
21 µL
2100 µL
21-30
wells
32 µL
3200 µL
31-40
wells
42 µL
4200 µL
Remove and discard Washing Buffer, and add 100 µl diluted conjugate to each well,
incubate at 30 °C, 10min and 550rpm.

Add 200 µl C5 Washing Buffer. Incubate at 30°C, 5 min and 550rpm.

Remove and discard Washing Buffer, add 100 µl of D1 substrate (precipitating dye, at 25°C,
see above) per well.

Incubate at 25°C, 10 min but do not shake !

Remove liquid completely.

The bottom of the ArrayStrips may be cautiously be cleaned with wipes, bubbles may be
removed by removing and adding D1.

Scan and process (see below).
Protocol for Eppendorf’s Thermomixer Comfort with microtitre plate adapter

Switch on the thermoshaker and let it pre-heat to 55°C.

Remove the ArrayStrip(s) from the pouch.
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
Insert the ArrayStrip(s) into the white frame. Assure the correct orientation (data matrix
barcode close to row (A) and proper fit.

Pre-wash the array in two steps:

First, PCR-grade distilled water, 200 µl per well, just pipette up and down four times,
remove and discard.

Second, C1 Hybridisation Buffer, 150 µl per well at 55°C, 5 min and 550 rpm

Add 90 µL of buffer C1 to each tube with (10 µL) labelled amplification product, mix gently

Remove the buffer from the well with the array and add the mixture of C1 and labelled
amplification product

Incubate at 55°C, 60 min and 550 rpm.

Remove liquid and add 200 µl C2 Washing Buffer. Incubate at 40°C, 12 min and 550 rpm,
remove and discard.

Add another 200 µl C2 Washing Buffer. Incubate at 40°C, 12 min and 550 rpm, remove and
discard.

Meanwhile, prepare conjugate: for each experiment add 1 µl conjugate 100xHRP to 100 µl
C4 Conjugation Buffer. This mixture is stable for around one working day at room
temperature; C3 is delivered with a surplus of 100%, C4 is delivered with a surplus of 200%.
Suggested pipetting scheme:
C3
C4

1
well
1.5 µL
150 µL
2-3
wells
3.5 µL
350 µL
4-6
wells
7 µL
700 µL
7-10
wells
11 µL
1100 µL
11-15
wells
16 µL
1600 µL
16-20
wells
21 µL
2100 µL
21-30
wells
32 µL
3200 µL
31-40
wells
42 µL
4200 µL
Remove and discard Washing Buffer, and add 100 µl diluted conjugate to each well,
incubate at 30°C, 10min and 550rpm.

Remove liquid and wash with 200 µl C5 Washing Buffer, just pipette up and down four
times, remove and discard.
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
Add another 200 µl C5 Washing Buffer, just pipette up and down four times.

Remove and discard Washing Buffer, add 100 µl of D1 substrate (precipitating dye, at 25°C,
see above) per well.

Incubate at 25°C, 10 min but do not shake !

Remove liquid completely.

The bottom of the ArrayStrips may be cautiously be cleaned with wipes, bubbles may be
removed by removing and adding D1.

Scan and process (see below).
Data Analysis
Starting the ArrayMate Reader
We recommend starting the ArrayMate Reader after having started the hybridisation; this
allows you to conveniently start the device and to import the worklist file (see below)
Please note that this is a short instruction only. For more detailed information please refer to
the ArrayMate User Manual.

Switch on the ArrayMate (main switch on the rear below the electric cable plug, operating
switch on the bottom/left corner of the front side).

Switch on the screen (switch right hand below the screen).

Log-in as “R&D User” (Research and Development User) for full access to test specific
software (a default password will be provided together with the ArrayMate device). If you
log-in as “User”, you will obtain only raw values, but no interpretation as
positives/negatives and no strain assignment. “Administrator” log-in will allow
manipulation of file folders and software; and this should be done only upon direct advice
of Clondiag´s IT team.
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
The user interface will be loaded, ArrayMate performs internal testing. This will require
slightly less than a minute.

Click on the icon “New Run” (left upper edge of the screen). A suggestion for a run name /
folder name for the new run appears in the top line of the screen). You may modify or
change the experiment name at your convenience.

Type in your operator ID (optional).

You may optionally enter a comment into the “memo” field.
Worklist
A “Worklist” file allows linking an identifier such as a laboratory/sample number to a position of
an array within the ArrayStrip. For privacy reasons, arrays should not be identified by patient
names. Worklists can be generated using spreadsheet software such as EXCEL (see below) but
must be saved in the *.txt file format that can be imported into the test specific ArrayMate
software. Do not use special characters (such as : ; ()[] / \ ä ü etc.).

Create a list with at least three columns that have headers written into the first line. The
following headers are obligatory (in this order): position / sample ID / assay ID (Table 1).

Positions are continuously numbered from 1 to, maximal, 96. Position 1 would correspond
to A1, 8 to H1, 9 to A2 and 96 to H12 (Table 2). Do not leave empty lines in the worklist. If
you use EXCEL, position numbers should be typed into column A.

Sample ID are strain/sample/laboratory numbers such as exported from your LIMS (or
assigned in any different way). Patient´s names should not be used as Sample IDs.

Assay IDs allow the system to identify the actual test and to correctly use information on
layout, spot number and identity etc. E. coli combined Assay has the Assay ID: 10313. Assay
ID numbers must not be confused as this could lead to errors or loss of data.

You may add further columns and headers with notes and comments at your convenience
Information from these columns will not appear on the result screens or the Test Report.

We recommend using a printout of the worklist as template for pipetting.
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
Safe the worklist as tab separated *.txt file on the memory stick provided together with the
ArrayMate.

To avoid confusion, make sure that worklists are named unambiguously or that worklists
from earlier experiments are deleted.
Table 1: Example worklist:
Position
1
2
3
4
5
6
7
8
sampleID
2013-12345
2013-12346
2013-12347
2013-12348
2013-12349
2013-12350
987654
E. coli EDL933
assayID
10313
10313
10313
10313
10313
10313
10313
10313
comment
Isolate referred from Dr. J. Doe.
Control strain
Table 2: Positions in the 96 well format
A
B
C
D
E
H
G
H
1
1
2
3
4
5
6
7
8
2
9
10
11
12
13
14
15
16
3
17
18
19
20
21
22
23
24
4
25
26
27
28
29
30
31
32
5
33
34
35
36
37
38
39
40
6
41
42
43
44
45
46
47
48
7
49
50
51
52
53
54
55
56
8
57
58
59
60
61
62
63
64
9
65
66
67
68
69
70
71
72
10
73
74
75
76
77
78
79
80
11
81
82
83
84
85
86
87
88
12
89
90
91
92
93
94
95
96
Data Acquisition in the ArrayMate Reader

Insert your memory stick containing the worklist. Use any of the USB ports down to the
right side of the ArrayMate.

Press the button:

Select your worklist (path: “My Computer/Removable Disk”).
; a folder selection dialog will open.
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
Open your selected worklist with “Enter” or the button “Open”.

Press the button:
(your imported worklist opens in a separate window). Proofread. If
the new window is empty or if it was the wrong worklist, repeat the import.

Press the button “OK”; the worklist window will close.

Leave the memory stick attached to the ArrayMate if you intend to export E. coli combined
Test Reports afterwards.

Press the button “next” (bottom/right on the screen; reader is opening).

Carefully insert the appropriate metallic adapter/frame into the ArrayMate. Do not apply
any strong force. Assure proper fit, otherwise the images may be out of focus.

Carefully insert the white frame with the array strips into the metallic adapter. Assure the
correct orientation (Position A1 in the frame next to the data matrix barcode on the
adapter) and proper fit, otherwise the images may be out of focus.
ArrayStrip frame with inserted
strips. Strips are inserted in
accordance to the worklist.
Please note: ArrayStrips must be clean. They should not contain any liquids by now. Barcodes
must be clean. There must be no lids on the wells that are to be analysed
(however, unused wells should remain capped).

Press the button “Next” (bottom/right on the screen; reader is closing, analysis program
starts, it takes ca. 2-10 min dependent on the number of strips; reader takes images and
automatically analyses the data). The progress of the reading is indicated by the following
symbols:
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photographed:
in analysis:
ready:

The reader indicates the end of the entire process with an acoustic signal (beep).

Press the button “Next” (bottom/right on the screen; reader is opening).

Remove the white frame with the ArrayStrip(s).

Press the button “Next” (bottom/right on the screen; reader is closing).
Results
On the left hand site of the screen you will see a list showing all runs stored on the ArrayMate´s
hard disk. A run contains the results from all arrays analysed together within one frame. If this
list is not visible:

Press the button “Archive” (left hand) and activate the Flag “Browse” (top left).
The runs are organised like folders in “Windows Explorer” and, by default, named according to
the date of acquisition.
Example: there is one reading in this archive:
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If you click on the plus symbol left on the run name, the folder opens and you will see a list of
the individual arrays alphabetically ordered by Sample ID.
Click on a Sample ID and the E. coli combined Test Report for this array is shown in the window
on the right:
Export of E. coli combined Test Reports
The generated result files in an html format will show information of all target genes. Possible
invalid controls that might display in this report will be explained below (see Troubleshooting).
Other files that are generated and that can be exported include

a *.txt file with the raw measurements,

an image file (*.bmp) with the actual photo of the array,
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
a second image file (*.png) in which the coordinate grid is superimposed and the
recognised spots are circled and

a *.xml files that contains the same information as the html result sheets for future
export into databases etc.
Please note: only complete runs can be exported. The export of individual E. coli combined
Test Reports is not possible

Right-Click on the reading (a menu appears with the option “Export Run Reports”).

Right-Click on “Export Run Reports” (a file browser opens).

Click on “My Computer”, then on “Removable Disk” and choose the folder where to store
or click on the button “Make New Folder” (on the bottom; a new folder icon appears).

Rename the new folder (e.g. with the experiment name or date).

Click on the “Ok” button (data are exported now into the new folder on your memory
stick).

Do NOT remove the memory stick as long as the hourglass symbol is visible.

Switch off the device by clicking on the “Power”-button (left/down on the screen):

Switch off the Screen. There is no need to physically switch off the ArrayMate.
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TROUBLESHOOTING
In case of trouble always make sure that reagents are within the recommended shelf-life and
stored in the appropriate way.
In case of trouble we are always happy to support. Please contact, cct.home@clondiag.com and
please include a description of the problem as well as the array images (*.bmp files) in
question.
Staining Control
A staining control is included to check whether possible problems originate from the
hybridisation or the staining procedure. If the staining control has “Failed” proceed as follows:
Horseradish peroxidase conjugate may have degraded during storage. Add 1µL buffer C3/C4 to
9 µL D1 (substrate). If the solution turns green within 3-5 seconds, the horseradish peroxidase
still has sufficient enzymatic activity.
Enzymatic reaction is inhibited by carryover of buffer C1. Ensure proper washing of the wells to
remove all of Buffer C1 prior to adding horseradish peroxidase conjugate.
If the Staining Control has “Passed”, refer to the following hints.
Image Quality
In case of poor image quality we recommend to re-check DNA quantity and quality first by
loading leftover DNA on an agarose gel.
In order to determine whether any problems originated from the DNA preparation, perform an
experiment with the CM / Control material. This is DNA from the reference strain E. coli EDL933
(GenBank accession number NC_002655.2) and should be identified by the assay as E. coli with
O:146 and H:20. If the control experiment yields a valid result and a correct identification, there
was probably an issue with DNA preparation. If the control experiment also fails, an error
affecting later steps or a degradation of reagents from later steps is likely.
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DNA Quality and RNA contamination control
The amount of DNA is crucial because of the linear kinetics of amplification (see Introduction).
DNA should be free of RNA, as free RNA reduces the efficiency of amplification and labelling by
effectively removing primer from the reaction mix due to competitive hybridisation. A260
readings will cover RNA and other contaminants as well. Therefore pure DNA preparations
without RNA contaminations are a prerequisite for proper DNA concentration measurement.
RNase treatment prior to A260 reading therefore is necessary (component A2 contains RNAse).
Additionally, the microarray includes probes as an internal control for RNA contamination. The
automatic software analysis will give a “failed” if a RNA contamination occurred while DNA
isolation.
DNA must be unfragmented, as fragmentation reduces the efficiency of amplification and
labelling due to the distance between primer and probe binding sites. DNA should for this
reason not be prepared by disrupting E. coli cells using bead beaters, ultrasonication or
aggressive chemicals such as in alkaline lysis protocols. We made good experiences with the
manual QIAGEN DNeasy kit and the Roche High Pure Kit.
DNA must be free of any traces of ethanol, as ethanol strongly influences the amplification. It is
possible to heat the sample prior to adding it to the labelling mix (5-10 minutes at 70°C).
Physical Damage to the Array
Scratching of the array surface with a pipette tip can lead to the damage of array spots that
prohibits the acquisition of a valid signal. In this case the respective marker is not assigned as
“negative”, but instead the message “none” appears next to the marker name.
Report Unavailable
If the ArrayMate indicates that no report is available for an array (or multiple arrays on one
strip), please check that the strip was positioned properly into the frame. Scratches or drops of
condensed water might render the barcode identifier unreadable, please wipe it carefully or try
to manually identify the test. If no obvious reason for the fault can be discovered, please
contact the technical service.
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ADDITIONAL INFORMATION
Warranty
Alere guarantees the performance as described in this user guide. Usage of the Assay was
successfully tested at ambient temperatures up to 37°C, a guarantee is limited to ambient
temperatures in the laboratory between 18°C and 28°C. Assay components comprise the arrays
and their caps, the Lysis Enhancer, the reagents for DNA labelling and for detection of labelled
DNA products on the array, the ArrayMate reader and its software. In case one of these
components fails within the expiry date due to other reason than misuse, contact Alere for
replacement or refund. Terms and conditions apply.
If you have any problem or question, please contact the technical service.
Disclaimer
This system is for research use only.
We do not accept any liability for damages caused by misuse. Misuse comprises, especially but
not exclusively, of a use of the system for the detection of resistance genes in order to predict
phenotypic antibiotic resistances or susceptibilities for the guidance of an antibiotic
chemotherapy.
Since resistances might be caused by genes or mutations not covered by this array or by hitherto
unknown genes or mutations, any antibiotic chemotherapy MUST be guided by phenotypic
susceptibility tests.
Furthermore, we do not accept any liability for damages caused by inappropriate use of the
device as a personal computer, for instance related to the use of additional software, to
network connections, or to a breach of privacy related to the storage of confidential
information (such as names of patients from whom E. coli was isolated) on its hard disk and/or
to the use of external storage devices that might be contaminated with spyware.
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Quality Control
Each batch is stringently tested with the use of standard E. coli DNA preparations for good
performance and correctness of results.
List of Components for Separate Order
If required, these reagents for the E. coli combined Assay may be ordered separately:
component
A1
A2
Ecoli-PM1
Category
Buffer
lyophylised enzymes
Labelling Primer
amount
cat#
storage
30 ml
245101000 18-28 °C
96 units
245102000 18-28 °C
50 µl/tube
on request
2-8 °C
B1+
name
Lysis Buffer
Lysis Enhancer
PrimerMix
E. coli_combined
(= E. coli_gesamt)
Labelling Buffer
buffered reagents
700 µl
245103000
2-8 °C
B2
C1
C2
C3
C4
C5
D1
CM
Labelling Enzyme
Hybridisation Buffer
Washing Buffer 1
HRP Conjugate 100x
Conjugate Buffer
Washing Buffer 2
HRP Substrate
Control Material
20 µl
30 ml
120 ml
200 µl
30 ml
120 ml
15 ml
30 µl
245104000
245105000
245106000
245107000
245108000
245109000
245110000
on request
2-8 °C
18-28 °C
18-28 °C
2-8 °C
18-28 °C
18-28 °C
2-8 °C
2-8 °C
ArrayStrips
E.coli-combined-AS-2
(=E.coli-gesamt-AS-2)
StripCaps
buffered enzyme
buffered reagents
buffer
buffered enzyme
buffered reagents
buffer
buffered reagents
E. coli EDL933 DNA (cDNA =
0.1-0.4 µg/µl)
plugged microarrays
12 St
240008929
15-28 °C
plasticware
24 St
245112000
15-28 °C
StripCaps
For pricing please contact us:
Legal Manufacturer
Alere Technologies GmbH
Loebstedter Str. 103-105
07749 Jena, Germany
Contact
If you require any further information on this product please contact: cct.home@clondiag.com
E. coli combined Assay
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LITERATURE
Anjum MF, Mafura M, Slickers P, Ballmer K, Kuhnert P, et al. (2007) Pathotyping Escherichia coli
by using miniaturized DNA microarrays. Appl Environ Microbiol 73: 5692-5697.
Ballmer K, Korczak BM, Kuhnert P, Slickers P, Ehricht R, et al. (2007) Fast DNA serotyping of
Escherichia coli by use of an oligonucleotide microarray. J Clin Microbiol 45: 370-379.
Geue L, Schares S, Mintel B, Conraths FJ, Muller E, et al. (2010) Rapid microarray-based
genotyping of enterohemorrhagic Escherichia coli serotype O156:H25/H-/Hnt isolates
from cattle and clonal relationship analysis. Appl Environ Microbiol 76: 5510-5519.
Korczak B, Frey J, Schrenzel J, Pluschke G, Pfister R, et al. (2005) Use of diagnostic microarrays
for determination of virulence gene patterns of Escherichia coli K1, a major cause of
neonatal meningitis. J Clin Microbiol 43: 1024-1031.
Monecke S, Mariani-Kurkdjian P, Bingen E, Weill FX, Baliere C, et al. (2011) Presence of
enterohemorrhagic Escherichia coli ST678/O104:H4 in France prior to 2011. Appl
Environ Microbiol 77: 8784-8786.
Schilling AK, Hotzel H, Methner U, Sprague LD, Schmoock G, et al. (2012) Zoonotic agents in
small ruminants kept on city farms in southern Germany. Appl Environ Microbiol 78:
3785-3793.
Wu G, Ehricht R, Mafura M, Stokes M, Smith N, et al. (2012) Escherichia coli isolates from
extraintestinal organs of livestock animals harbour diverse virulence genes and belong
to multiple genetic lineages. Vet Microbiol 160: 197-206.
UPDATES & SOFTWARE
Notifications on database/software updates and freeware tools can be found at :
http://alere-technologies.com/en/science-technologies/publications/downloads.html.
and/or http://alere-technologies.com/en/news.html.
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APPENDIX 1 – Flow charts
The figures summarise the test procedure. However, please refer to the text section of this user
guide at any step of the test protocol for further important details.
Protocol : Quantifoil BioShake iQ
processing
time
handsontime
Grow CLONAL E. coli isolate
(not part of the kit)
over
night
5 min
isolate genomic DNA
(not part of the kit)
3-4 h
40 min
2h
5 min
2 min
2 min
2 min
2 min
hybridise; 50 °C, 550 rpm; 60 min
60 min
0 min
discard labeled DNA;
incubate twice in 200 µl Buffer C2; 45 °C, 550 rpm, 10 min;
prepare C3/C4-conjugate (C3:C4=1:100), preheat Substrat D1 (25°C)
20 min
5 min
discard Buffer C2;
incubate in 100 µl C3 /C4-conjugate; 30 °C, 550 rpm, 10 min
10 min
2 min
discard C3/C4-conjugate;
incubate once in 200 µl Buffer C5; 30 °C, 550 rpm, 5 min
5 min
2 min
discard Buffer C5;
incubate with 100 µl Substrate D1; 25 °C, 10 min
10 min
2 min
discard Substrate D1; analyse (ArrayMate)
15 min
prepare ArraysStripes
rinse ArrayStripes
200 µl water; 50 °C, 550 rpm, 5 min
discard water;
150 µl Buffer C1; 50 °C, 550 rpm, 5 min
discard C1, process promptly
prepare DNA
label RNA free DNA in thermocycler
5 µl DNA (cDNA = 0.1 - 0.4 µg/µl)
plus MM (3.9 µL B1+ + 0.1 µL B2 +
1 µl PM Ecoli-PM1)
preparing labeled DNA
to 10 µL of labeled DNA add 90 µL
of Buffer C1
transfer 100 µl labeled DNA to ArrayStripes
Barcode
MM - MasterMix
PM - PrimerMix
a) with
Label here
total time requirement : over night
+ 7-8h
10 min
app. 120 min
heating block for microtitre plates
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Protocol : Eppendorf Thermoshaker a)
processing
time
handsontime
Grow CLONAL E. coli isolate
(not part of the kit)
over
night
5 min
isolate genomic DNA
(not part of the kit)
3-4 h
40 min
2h
5 min
10 min
5 min
2 min
2 min
hybridise; 55 °C, 550 rpm; 60 min
60 min
0 min
discard labeled DNA;
incubate twice in 200 µl Buffer C2; 40 °C, 550 rpm, 12 min;
prepare C3/C4-conjugate (C3:C4=1:100), preheat Substrat D1 (25°C)
25 min
5 min
discard Buffer C2;
incubate in 100 µl C3 /C4-conjugate; 30 °C, 550 rpm, 10 min
10 min
2 min
discard C3/C4-conjugate;
incubate once in 200 µl Buffer C5; 30 °C, 550 rpm, 5 min
5 min
2 min
discard Buffer C5;
incubate with 100 µl Substrate D1; 25 °C, 10 min
10 min
1 min
discard Substrate D1; analyse (ArrayMate)
15 min
10 min
prepare ArraysStripes
prepare DNA
rinse ArrayStripes
200 µl water; pipette up and down (4x)
label RNA free DNA in thermocycler
5 µl DNA (cDNA = 0.1 - 0.4 µg/µl)
plus MM (3.9 µL B1+ + 0.1 µL B2 +
1 µl PM Ecoli-PM1)
discard water;
150 µl Buffer C1; 55 °C, 550 rpm, 5 min
discard C1, process promptly
preparing labeled DNA
to 10 µL of labeled DNA add 90 µL
of Buffer C1
transfer 100 µl labeled DNA to ArrayStripes
Barcode
MM - MasterMix
PM - PrimerMix
a) with
Label here
total time requirement : over night
+ 7-8h
app. 120 min
heating block for microtitre plates
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APPENDIX 2 – PROBE TO TARGET TABLE
Target genes
Probes
Function
Family and Species Marker
gapA
prob_gapA_611
ihfA
prob_ihfA_611
gad
gad_10
dnaE
hp_dnaE_612, hp_dnaE_613
rrs
hp_rrs_611, hp_rrs_612
glyceraldehyde 3-phosphate dehydrogenase A
(CP000468.1, locus tag APECO1_847)- genetic marker
for family Enterobacteriaceae
integration host factor subunit alpha (U00096.2)genetic marker for family Enterobacteriaceae
glutamate decarboxylase (AE014075.1, locus tag
c4328) - genetic marker for genus Escherichia/Shigella
DNA polymerase III subunit alpha - genetic marker for
species Escherichia spec. and Shigella spec.
(U00096.2)
16S rRNA - genetic marker for species E. coli
(U00096.2)
O4
wzy-O4_11
O-antigen - O4 (U39042.1)
O6
wzx-O6_11, wzy-O6_11
O-antigen - O6 (AJ426045.2)
O7
wzx-O7_11, wzy-O7_11
O-antigen - O7 (AB490074.1)
O8
wzx-O8_11, wzy-O8_11
O9
wzx-O9_11, wzy-O9_11
O-antigen - O8 (AF013583.1)
O-antigen - O9 (wbdA_O9: D43637.1, wzx_O9:
AF104912.2, wzy_09: AB031867.1)
O15
wzx-O15_11, wzy-O15_11
O-antigen - O15 (CP002291.1)
O26
wzx-O26_11, wzy-O26_11
O-antigen - O26 (AF529080.1)
O52
wzm-O52_11
O-antigen - O52 (AY528413.1)
O53
wzy-O53_11
O-antigen - O53 (AF402312.1)
O55
wzx-O55_11, wzy-O55_11
O-antigen - O55 (AF461121.1)
O79
wbdU-O79_11, wzx-O79_11
O-antigen - O79 (EU294162.1)
O86
wzx-O86_11, wzy-O86_11
O-antigen - O86 (AY670704.1)
O91
wzx-O91_11, wzy-O91_11
O-antigen - O91 (AY035396.1)
O101
wz-O101_11, wbdA-O9_11
O-antigen - O101 (X59852.1)
O103
wzx-O103_11, wzy-O103_11
O-antigen - O103 (AY532664.1)
O104
O111
wzx-O104_11, wzy-O104_11
O-antigen - O104 (AF361371.1)
wbdH-O111_11, wbdM-O111_11,
wzx-O111_11, wzy-O111_11
O-antigen - O111 (AF078736.1)
O113
wzx-O113_11, wzy-O113_11
O-antigen - O113 (AF172324.1)
O114
wzx-O114_11, wzy-O114_11
O-antigen - O114 (AY573377.1)
O121
wzx-O121_11, wzy-O121_11
O-antigen - O121 (AY208937.1)
O128
wzx-O128_11, wzy-O128_11
O-antigen - O128 (AY217096.1)
O157
rfbE-O157_11, wzx-O157_11
O-antigen - O157 (AB008676.1)
O172
wzx-O172_11, wzy-O172_11
O-antigen - O172 (AY545992.1)
O-serotyping
E. coli combined Assay
User Guide 13-11-11-001-V1
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H-serotyping
fliC H01
fliC-H01_11, fliC-H01_12
flagellin C H-antigen H01 (AE014075.1)
fliC H02
fliC-H02_11
flkA H03
flkA-H03_11
flagellin C H-antigen H02 (AF543692.1)
flagellin A H-antigen H03 (AB128916.1) (Note: If
repressor FljA "positive", the gene fliC H16 is
repressed; H-phenotype is H03)
fliC H04
fliC-H04_11
flagellin C H-antigen H04 (AY249989.1)
fliC H05
fliC-H05_11
flagellin C H-antigen H05 (AY249990.1)
fliC H06
fliC-H06_11
flagellin C H-antigen H06 (AY249991.1)
fliC H07
fliC-H07_11, fliC-H07_12
flagellin C H-antigen H07 (AB028474.1)
fliC H08
fliC-H08_11
flagellin C H-antigen H08 (AJ884571.1)
fliC H09
fliC-H09_11
flagellin C H-antigen H09 (AY249994.1)
fliC H10
fliC-H10_11
flagellin C H-antigen H10 (AY249995.1)
fliC H11
fliC-H11_11
flagellin C H-antigen H11 (AY337465.1)
fliC H12
fliC-H12_11
flagellin C H-antigen H12 (AY249997.1)
fliC H14
fliC-H14_11
flagellin C H-antigen H14 (AY249998.1)
fliC H15
fliC-H15_11
fliC H16
fliC-H16_11
flagellin C H-antigen H15 (AY249999.1)
flagellin C H-antigen H16 (AB128919.1), Note: If
repressor fljA "positive", fliC H16 might be repressed,
H-phenotype is than determined by flkA H03.
fliC H18
fliC-H18_11
flagellin C H-antigen H18 (AY250001.1)
fliC H19
fliC-H19_11
flagellin C H-antigen H19 (AY250002.1)
fliC H20
fliC-H20_11
fliC H21
fliC-H21_11
flagellin C H-antigen H20 (AY250003.1)
flagellin C H-antigen H21 (DQ862122.1), Note: If
repressor fljA "positive", fliC H21 might be repressed,
H-phenotype is than determined by flmA H54.
fliC H23
fliC-H23_11
flagellin C H-antigen H23 (AY250005.1)
fliC H24
fliC-H24_11
flagellin C H-antigen H24 (AY250006.1)
fliC H25
fliC-H25_11
flagellin C H-antigen H25 (ADUP01000024.1)
fliC H26
fliC-H26_11
flagellin C H-antigen H26 (AY250008.1)
fliC H27
fliC-H27_11
flagellin C H-antigen H27 (CU928162.2)
fliC H28
fliC-H28_11
flagellin C H-antigen H28 (AY250010.1)
fliC H29
fliC-H29_11
flagellin C H-antigen H29 (AY250012.1)
fliC H30
fliC-H30_11
flagellin C H-antigen H30 (AY250011.1)
fliC H31
fliC-H31_11
flagellin C H-antigen H31 (AY250013.1)
fliC H32
fliC-H32_11
flagellin C H-antigen H32 (AY250014.1)
fliC H33
fliC-H33_11
flagellin C H-antigen H33 (AY250015.1)
fliC H34
fliC-H34_11
flagellin C H-antigen H34 (AY250016.1)
fliC H37
fliC-H37_11
flagellin C H-antigen H37 (AY250017.1)
fliC H38
fliC-H38_11
flagellin C H-antigen H38 (AY250018.1)
fliC H39
fliC-H39_11
fliC H40
fl-H40_11
flagellin C H-antigen H39 (AY250019.1)
flagellin C H-antigen H40 (AJ865464.1), Note: If
repressor fljA "positive", fliC H40 might be repressed,
H-phenotype is than determined by flkA H53.
E. coli combined Assay
User Guide 13-11-11-001-V1
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fliC H41
fliC-H41_11
flagellin C H-antigen H41 (AY250020.1)
fliC H42
fliC-H42_11
flagellin C H-antigen H42 (AY250021.1)
fliC H43
fliC-H43_11
flagellin C H-antigen H43 (AY250022.1)
fliC H45
fliC-H45_11
flagellin C H-antigen H45 (AY250023.1)
fliC H46
fliC-H46_11,fliC-H46_12
flagellin C H-antigen H46 (AY250024.1)
fliC H48
fliC-H48_11
flagellin C H-antigen H48 (AY250025.1)
fliC H49
fliC-H49_11
flagellin C H-antigen H49 (AY250026.1)
fliC H51
fliC-H51_11
flagellin C H-antigen H51 (AY250027.1)
fliC H52
fliC-H52_11
flkA H53
flkA-H53_11
flmA H54
flmA-H54_11
flagellin C H-antigen H52 (AY250028.1)
flagellin A H-antigen H53 (AB128917.1) (Note: If
repressor FljA "positive", the gene fliC H40 is
repressed; H-phenotype is H53)
flagellin A H-antigen H54 (AB128918.1) (Note: If
repressor FljA "positive", the gene fliC H21 is
repressed; H-phenotype is H54)
fliC H56
fliC-H56_11
flagellin C H-antigen H56 (AY250029.1)
H-serotyping (additional marker)
fliC-5p_11, fliC-5p_12, fliC-5p_13,
fliC Marker
fliC-5p_14, fliC-5p_15
fljA Repressor
fljA_11
fliC marker - consensus sequence for all fliC genes
FljA represses the expression of fliC genes, if
"positive" the genes flkA (H03 or H53) or flmA (H54)
represent the H-serotype (AB128916.1)
fliC non-motile
fl-H-NM_11
flagellin C from non-motile isolates (AY337480.1)
streptogramin A resistance
vatE
hp_vatE_611, hp_vatE_612
acetyltransferase;streptogramin A acetyltransferase;
associated with resistance to streptogramin A
(AF242872.1)
aminoglycoside resistance
strA, strB
prob_strA_611, prob_strB_611
aac_aph
aac3
hp_aac_aph_611
hp_aac3_611, hp_aac3_612,
hp_aac3_613, hp_aac3_614,
hp_aac3_615
aac3Ia
prob_aac3Ia_1
aac3IVa
prob_aac3IVa_1
hp_aac6_611, hp_aac6_612,
hp_aac6_613, hp_aac6_614,
hp_aac6_616, hp_aac6_617,
hp_aac6_618
aac6
E. coli combined Assay
User Guide 13-11-11-001-V1
aminoglycoside-3''-phosphotransferase (locus A) and
aminoglycoside-6''-phosphotransferase; associated
with resistance to streptomycin (EF090911.1)
3-N-aminoglycoside acetyltransferase; associated with
resistance to gentamycin (AE017171.1)
3-N-aminoglycoside acetyltransferase; associated with
resistance to gentamycin (U90945.1)
3-N-aminoglycoside acetyltransferase; associated with
resistance to astromicin; gentamicin; sisomicin
(U90945.1)
3-N-aminoglycoside acetyltransferase; associated
with resistance to apramycin; dibekacin; gentamicin;
netilmicin; sisomicin; tobramycin (EU784152.1)
aminoglycoside 6'-N-acetyltransferase, associated
with resistance to amikacin; dibekacin; isepamicin;
netilmicin; sisomicin; tobramycin (AF162771.1)
33
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aac6Ib
prob_aac6Ib_1
aadA1
prob_aadA1_1
aadA2
prob_aadA2_1
aadA4
prob_aadA4_1
aadB
hp_aadB_611, hp_aadB-2_611
ant2
aphA
prob_ant2Ia_1
hp_aphA_611, hp_aphAvar4_611, hp_aphA-var5_611
grm
hp_grm_611
armA
hp_armA_611
rmtA
hp_rmtA_611
rmtB
hp_rmtB_611
rmtC
hp_rmtC_611
rmtD
hp_rmtD_611
npmA
hp_npmA_611
aminoglycoside 6'-N-acetyltransferase; associated
with resistance to streptomycin, spectinomycin
(M21682.1)
aminoglycoside adenyltransferase; associated with
resistance to streptomycin, spectinomycin
(EU704128.1)
aminoglycoside adenyltransferase; associated with
resistance to streptomycin, spectinomycin
(EU704128.1)
aminoglycoside adenyltransferase; associated with
resistance to streptomycin, spectinomycin (Z50802.3)
2''-aminoglycoside nucleotidyltransferase (L06418.4)
aminoglycoside (2'') adenylyltransferase; associated
with resistance to dibekacin; gentamicin; kanamycin;
sisomicin; tobramycin (L06418.4)
aminoglycoside 3'-phosphotransferase; kanamycin
resistance protein (AY260546.3)
16S rRNA methylase, associated with gentamicin
resistance (M55521.1)
16S rRNA methylase, associated with aminoglycoside
resistance (AB117519.1)
16S rRNA methylase, associated with aminoglycoside
resistance (AB083212.2)
16S rRNA methylase, associated with aminoglycoside
resistance (DQ345788.1)
16S rRNA methylase, associated with aminoglycoside
resistance (AB194779.2)
16S rRNA methylase, associated with aminoglycoside
resistance (DQ914960.2)
16S rRNA methylase, associated with aminoglycoside
resistance (AB261016.1)
beta lactam resistance
blaACC
prob_acc2_11, prob_acc1_11
blaACT
prob_act1_11
blaCMY
hp_blaCMY_611,
hp_blaCMY_612, prob_cmy_11
blaKHM
blaMOX-CMY9
blaCTX-M1, blaCTXM15
blaCTX-M2, blaCTXM8, blaCTX-M26
ctxM9
class C beta-lactamase blaACC-1/blaACC2(EF554600.1)
class C beta-lactamase blaACT-1(U58495.2)
consensus sequence for blaCMY-13, blaCMY-2,
blaCMY-24, blaCMY-35, blaCMY-CFE1, blaCMY-CFE2
(Citrobacter spp.), blaCMY-Cmur (Citrobacter
murliniae), blaCMY-Cwer (Citrobacter werkmanii),
blaCMY-Cyou (Citrobacter youngae), blaCMY-HG3,
blaCMY-HG4
hp_blaKHM-1_611
hp_blaMOX-CMY9_611,
hp_blaMOX-CMY9_612,
hp_blaMOX-CMY9_613
class B metallo beta-lactamase (AB364006.1)
class C extended-spectrum beta-lactamase precursor,
associated with resistance to cephalosporins
(AF381617.1)
class A extended-spectrum-beta-lactamase
prob_ctxM1_11, prob_ctxM1_12 (X92506.1), including blaCTX-M15 (HQ202266.1)
prob_ctxM2_11,
class A extended-spectrum-beta-lactamase
prob_ctxM26_11, prob_ctxM8_11 (AM040709.1, AF518567.2, AY750914.2)
prob_ctxM9_11, prob_ctxM9_12
E. coli combined Assay
User Guide 13-11-11-001-V1
class A beta-lactamase (AF174129.3)
34
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blaDHA-1
prob_dha1_1
class C beta-lactamase (EF406115.1)
blaFOX
prob_fox_11, prob_mox_1mm
class C beta-lactamase blaFOX (consensus)
blaGES-1
hp_ges-1_611
class A beta-lactamase (AY219651.1)
blaGIM-1
hp_gim1_611
blaIMI-3
blaIMP
hp_imi3_611
hp_imp_611, hp_imp_612,
hp_imp_613, hp_imp_614,
hp_imp_615, hp_imp_616,
hp_imp_617
class B metallo beta-lactamase (AJ620678.1 )
class A metallo beta-lactamase - carbapenemase,
associated with imipenem resistance (AY780889.1)
blaKPC-4
hp_kpc4_611
class A beta lactmase - carbapenemase (EU447304.1.)
blaLAP-1
hp_lap1_611
class A beta-lactamase (EF026092.1)
blaLEN-1
prob_len1_11
class A beta-lactamase (AY743416.1)
blaMOX
prob_mox_1pm
blaOXA-1
prob_oxa1_21
blaOXA-2
prob_oxa2_11
blaOXA-7
prob_oxa7_11
blaOXA-9
prob_oxa9_11
class C beta-lactamase blaMOX (consensus)
oxacillinase - class D beta-lactamase blaOXA-1
(AY458016.1)
oxacillinase - class D beta-lactamases blaOXA2/blaOXA-15 (U63835.1)
oxacillinase - class D beta-lactamase blaOXA-7
(AY866525.1)
oxacillinase - class D beta-lactamase blaOXA-9
(M55547.1)
blaOXA-23
hp_oxa_611
oxacillinase - class D beta-lactamase (AJ132105.1)
blaOXA-40
hp_oxa_612
blaOXA-48
hp_oxa_613
oxacillinase - class D beta-lactamase (AF509241.1)
oxacillinase - class D carbapenem-hydrolyzing betalactamase (AY236073.2 )
blaOXA-51
hp_oxa_614
oxacillinase - class D beta-lactamase (CP000863.1)
blaOXA-54
hp_oxa_615
oxacillinase - class D beta-lactamase (AY500137.1)
blaOXA-55
hp_oxa_616
oxacillinase - class D beta-lactamase (AY343493.1)
blaOXA-58
hp_oxa_617
oxacillinase - class D beta-lactamase (AY665723.1)
blaOXA-60
hp_oxa_618
blaPER-1
hp_per1_611
blaPER-2
blaPSE-1
hp_per2_611, prob_per2_1
prob_pse1_1pm,
prob_pse1_1mm
oxacillinase - class D beta-lactamase (AF525303.2)
class A beta-lactamase PER-1; extended-spectrum
beta-lactamase (Z21957.1)
class A beta-lactamase PER-2; extended-spectrum
beta-lactamase (X93314.1)
blaSHF-1
hp_sfh1_611
blaSHV
prob_shv1_11
blaSME-1
hp_sme1_611
blaSPM-1
hp_spm1_611
blaTEM
prob_tem1_1
E. coli combined Assay
User Guide 13-11-11-001-V1
class B metallo beta-lactamase (consensus),
associated with imipenem resistance
carbenicillinase (Z18955.1)
class B metallo beta-lactamase (AF197943.1)
class A beta-lactamase - consensus sequence for
blaSHV genes, including extended-spectrum betalactamases
carbapenem hydrolysing beta-lactamase (Serratia
marcescens)(Z28968.1)
metallo beta-lactamase, carbapenemase
(AY341249.1)
class A beta-lactamase - consensus sequence for
blaTEM genes, including extended-spectrum betalactamases
35
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blaVEB-1
extended-spectrum beta-lactamase (AF010416.1)
blaVIM
hp_veb1_611
hp_vim_611, hp_vim_612,
hp_vim_613
ble
hp_ble_611
bleomycin resistance (X01702.1)
class B metallo beta-lactamase (Y18050.2)
chloramphenicol resistance
chloramphenicol acetyltransferase (group A)
(V00622.1)
chloramphenicol acetyltransferase (group B)
(AJ009818.1)
catA1
prob_catA1_11
catB3
prob_catB3_11
catB8
prob_catB8_12
catIII
prob_catIII_1
chloramphenicol acetyltransferase (AF227506.1)
chloramphenicol acetyltransferase (type III)
(AJ249249.1)
cmlA1
prob_cmlA1_11
chloramphenicol transporter (EF113389.1)
floR
prob_floR_11
florfenicol export protein (AF252855.1)
erythromycin resistance
ereA
hp_ereA_611, hp_ereA_612
type I erythromycin resistance (AY183453.1)
ereB
hp_ereB_611, hp_ereB_612
type II erythromycin resistance (AB207867.1)
macrolide resistance
ermB
mphA
hp_ermB_611, hp_ermB_612
hp_mph2_611, hp_mphA_611,
hp_mphB_611, hp_mphBM_611,
hp_mphD_611,
mrx
hp_mrx_611
rRNA adenine N-6-methyltransferase, lincosamide and
streptogramin B resistance protein (AB089505.1)
macrolide 2'-phosphotransferase (EF102240.1)
member of macrolide inactivation gene cluster mphAmrx-mphR (AB038042.1)
quinolione resistance
qepA
hp_qepA_611
qnrA1
prob_qnr_11, prob_qnr_12
qnrB
prob_qnrB_11, prob_qnrB_12
qnrD
hp_qnrD_611
qnrS
prob_qnrS_11
QepA - fluoroquinolone/quinolone efflux pump
(AM886293.1)
quinolone or fluoroquinolone resistance protein
(AY931018.1)
quinolone or fluoroquinolone resistance protein
(AB281054.1)
quinolone or fluoroquinolone resistance protein
(FJ228229.1)
quinolone or fluoroquinolone resistance protein
(AM234722.1)
hp_arr-1_611, hp_arr-4_611,
hp_arr-5_611, hp_arr-6_611
ADP-ribosyltransferase, associated with resistance to
rifampin (AF078527.1)
rifampin resistance
arr
streptomycin resistance
sph
hp_sph_611
streptomycin 3''-phosphotransferase (U00004.1)
streptomycin resistance
sul1
prob_sul1_11
dihydropteroate synthetase type 1 (AJ698325.1)
sul2
prob_sul2_11
dihydropteroate synthetase type 2 (DQ464881.1)
sul3
prob_sul3_11
dihydropteroate synthetase type 3 (AJ459418.2)
E. coli combined Assay
User Guide 13-11-11-001-V1
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tetracycline resistance
tet37
hp_tet37_611
tetA
prob_tetA_11
tetracycline resistance protein (AF540889.1)
tetracycline resistance protein A, tetracycline efflux
protein (CP000971.1)
tetB
prob_tetB_11
tetracycline resistance protein A, class B (V00611.1)
tetC
prob_tetC_11
tetracycline resistance protein A, class C (EU751612.1)
tetD
prob_tetD_1
tetracycline resistance protein A, class D (X65876.1)
tetE
prob_tetE_11
tetracycline resistance protein A, class E (L06940.1)
tetG
prob_tetG_11, prob_tetG_12
tetracycline resistance protein A, class G (AF261825.2)
tetX
hp_tetX_611
tetracycline resistance protein (M37699.1)
trimethoprim resistance
dfrA1
prob_dfrA1_21, prob_dfrA1_22
dihydrofolate reductase type 1 (AJ884723.1)
dfrA5
prob_dfrV_21
dfrA7
prob_dfrA7_11, prob_dfrA7_12
dihydrofolate reductase type 5 (AB188269.1)
dihydrofolate reductase type 7 (AB161450.1,
AM237806.1)
dfrA12
prob_dfr12_11
dfrA13
prob_dfr13_11
dihydrofolate reductase type 12 (AB154407.1)
dihydrofolate reductase type 13 (synonym A21)
(Z50802.3)
dfrA14
prob_dfrA14_21
dihydrofolate reductase type 14 (AJ313522.1)
dfrA15
prob_dfrA15_1
dihydrofolate reductase type 15 (Z83311.1)
dfrA17
prob_dfrA17_11
dihydrofolate reductase type 17 (AF169041.1)
dfrA19
prob_dfrA19_1
dihydrofolate reductase type 19 (AJ310778.1)
virulence factor - adhesins
eae_consensus_10,
eae_consensus_20,
eae_consensus_30,
eae - consensus
eae_consensus_40
efa1
hp_efa1_611
an outer membrane protein important for the
attachment to host cells; pathogenesis factor
(M58154.1)
lymphocyte inhibitory factor A - adherence factor
(AF159462.2)
fasA
fasA_10
adhesin - fimbrial major subunit (M35257.1)
fedA
fedA_10
adhesin - fimbrial major subunit (M61713.1)
fedF
fedF_10
adhesin - fimbrial protein (Z26520.1)
fim41a
fim41a_10
adhesin - fimbrial protein (X14354.1)
iha
hp_iha_611
nfaE
nfaE_10
espB_O157
espB_O157_20
espB_O26
espB_O26_40
adherence-conferring protein (BA000007.2)
chaperone protein - required for the expression of
aggregative adherence fimbria II (S61968.1)
EspB - protein (type III secretion system; O157:H7)
(BA000007.2)
EspB - protein (type III secretion system, 26:H- and
O15:H-) (AJ287768.1)
saa
hp_saa_611
STEC autoagglutinating adhesin (AF399919.3)
E. coli combined Assay
User Guide 13-11-11-001-V1
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virulence factor - fimbrae
bfpA
bfpA_10
BfpA protein - essential for apoptosis signalling
(AB024946.1)
cfaC
cfa_c_10
outer membrane usher protein (M55661.1)
cofA
cofA_10
major pilin subunit - CFA/III pilin (D37957.1)
f17-A
f17-A_40, f17-A_50, f17-A_60
major fimbrial subunit protein (L77091.1)
f17-G
f17-G_20
major fimbrial subunit protein (pilin G) (L43372.1)
fanA
fanA_10
regulatory protein (X05797.1)
K88ab
K88ab_10
major subunit of K88 fimbriae (V00292.1)
lngA
lngA_20
longus pilus structural subunit (EF595770.1)
lpfA
hp_lpfA_611
major fimbrial subunit (AY057066.1)
perA
perA_10, perA_20
prfB
prfB_30
transcriptional activator (AF255772.1)
major pilu subunit operon regulatory protein
(X76613.1)
sfaS
sfaS_10
adhesin - minor Shigella fimbriae subunit (X16664.4)
virulence factor - secretion systems
cif
hp_cif_611
espA_C_rodentium
espA
hp_espA_Crod_611
hp_espA_O103H2_611,
hp_espA_O119H6_611,
hp_espA_O127H7_611,
hp_espA_O157H11_611,
hp_espA_O49H12_611,
hp_espA_O55H7_611,
hp_espA_O8_611
espC
hp_espC_611
espF
hp_espF_611, hp_espF_612
espF_C_rodentium
espF_O103H2
hp_espF_Crod_611
hp_espF_O103H2_611,
hp_espF_O103H2_612
espI
hp_espI_611
espJ
hp_espJ_611, hp_espJ_612
etpD
nleA
hp_etpD_611
hp_nleA_611, hp_nleA_612,
hp_nleA_613, hp_nleA_614
nleB
hp_nleB_611
nleB O157:H7
hp_nleB_O157H7_611
E. coli combined Assay
User Guide 13-11-11-001-V1
cell cycle inhibiting factor (type III secretion system)
(AY128535.1)
EspA - protein (type III secretion system), associated
with Citrobacter rodentium (AF311901.1)
EspA - protein (type III secretion system) (AF054421.1)
EspC - extracellular serine protease (type III secretion
system) (AF297061.1)
EspF - effector protein (type III secretion system)
(AE005174.2)
EspF - effector protein (type III secretion system)
(AF311901.1)
EspF - effector protein (type III secretion system)
(AJ277443.1)
EspI - non-LEE encoded effector protein (LEE - EPEC
Locus of Enterocyte Effacement) (AJ278144.1)
EspJ - non-LEE encoded effector protein (LEE - EPEC
Locus of Enterocyte Effacement) (AE005174.2)
EtpD - type II secretion pathway related protein
(AF074613.1)
NleA - non-LEE-encoded effector protein A (type III
secretion system) (AM421997.1)
NleB - non-LEE-encoded effector protein B (type III
secretion system (BA000007.2)
NleB - non-LEE-encoded effector protein B (type III
secretion system), associated with serotype O157:H7
(BA000007.2)
38
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nleB Salmonella
hp_nleB_Styp_611
nleC
hp_nleC_611
tccP
hp_tccP_611, hp_tccP_612
NleB - non-LEE-encoded effector protein B (type III
secretion system), associated with Salmonella
enterica (AE008894.1)
NleC - non-LEE-encoded effector protein C (type III
secretion system) (AY485823.1)
Tir - cytoskeleton coupling protein (type III secretion
system) (AB275113.1)
virulence factor - SPATE (serin protease autotransporters)
EspP - serine protease autotransporter of
Enterobacteriaceae (SPATE) (AF074613.1)
Pic - serin protease autotransporter of
Enterobacteriaceae (SPATE) (U35656.1)
RpeA - serin protease autotransporter of
Enterobacteriaceae (SPATE) (AY552473.1)
SepA - serine protease autotransporter of
Enterobacteriaceae (SPATE) (AY604009.1)
SigA - serine protease autotransporter of
Enterobacteriaceae (SPATE) (AF200692.2)
Tsh - hemoglobin-binding protease (SPATE)
(AJ223631.1)
espP
hp_espP_611
pic
hp_pic_611
rpeA
hp_rpeA_611
sepA
hp_sepA_611
sigA
hp_sigA_611
tsh
hp_tsh_611
vat
hp_vat_611
eaaA
hp_eaaA_611
eatA
hp_eatA_611
epeA
hp_epeA_611
Vat - haemoglobin protease (SPATE) (AF242872.1)
EaaA - serine protease autotransporter of
Enterobacteriaceae (SPATE)(AF151674.1 )
EatA - serine protease autotransporter of
Enterobacteriaceae (SPATE) (AY163491.2)
EpeA - serine protease autotransporter of
Enterobacteriaceae (SPATE) (AY258503.2)
astA
astA_consens_10
heat stable enterotoxin (consensus sequence)
virulence factor - SPATE (serin protease autotransporters)
cba
cba_10
colicin B activity protein (M16816.1)
ccl
ccl_10
colicin activity protein (AF540491.1 )
cdtB
cdtB_40, cdtB_50, cdtB_60
cytolethal distending toxin subunit B (AJ508930.1)
celB
celb_10
colicin lysis protein (X03632.1)
cma
cma_20
colicin M activity protein (CP000971.1)
cnf1
cnf1_20
cytotoxic necrotizing factor type 1 (CP000243.1)
hlyA
hlyA_20
hemolysin A (AB011549.2)
hlyE
hlyE_10
hemolysin E (AF052225.1)
ipaD
ipaD_10
IpaD - invasin (CP000035.1)
ipaH
ipaH9.8_20
IpaH - invasion plasmid antigen (CP000039.1)
ltcA
ltcA_20
heat labile enterotoxin subunit A (AB011677.1)
mchB
mchB_10
microcin H47 activity protein (AJ515252.1)
mchC
mchC_20
member of the microcin operon (AJ515252.1)
mchF
mchF_10
putative microcin L transport protein (AJ515252.1)
mcmA
mcmA_10
microcin M truncated protein (AJ515252.1)
pet
pet_20
enterotoxin (SPATE) (AF056581.1)
sat
hp_sat_611
Sat serine protease (SPATE) (AJ586888.1)
E. coli combined Assay
User Guide 13-11-11-001-V1
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senB
senB_20
enterotoxin (Z54195.1)
sta1
sta1_110
heat stable enterotoxin I (AJ555214.1)
sta2
sta2_210
stb
stb_10
heat stable enterotoxin II (CP000795.1)
heat stable enterotoxin Stb - enterotoxin B
(M35729.1)
stx1 - consensus
shiga toxin 1
stx2 - consensus
stx1A_10
hp_stxA2_611, hp_stxA2_613,
hp_stxA2_614, hp_stxA2_615,
hp_stxA2_616, hp_stxA2_617,
hp_stxA2_618, stx2A_10,
hp_stxB2_612, hp_stxB2_613,
hp_stxB2_614, hp_stxB2_615
stx2b
hp_stxB2_612
shiga toxin 2 variant b (AB012101.1)
stx2e
hp_stxA2_616
shiga toxin 2 variant e (X81415.1)
stx2f
hp_stxA2_611, hp_stxA2_613
shiga toxin 2 variant f (AJ270998.1)
stx2g
hp_stxA2_617
hp_stxA2_614, hp_stxA2_618,
hp_stxB2_614, stx2A_10
shiga toxin 2 variant g (AJ966782.1)
shiga toxin 2 variant a, shiga toxin 2 variant c or shiga
toxin 2 variant d (X61283.1)
subtilase cytotoxin, subunit A (AF399919.3)
toxB
hp_subA_611
hp_toxB_611, hp_toxB_612,
hp_toxB_613
virF
virF_20
stx2a,c,d
subA
shiga toxin 2 (NOTE: variant classification by Scheutz
et al. 2012)
cytotoxin B (AB011549.2)
transcriptional regulator - required for transcription of
virB and icsA (AF348706.1)
virulence factor - miscellaneous
hemL
hp_hemL_612
glutamate-1-semialdehyde aminotransferase
(U00096.2)
intI1
prob_intI1_1
class 1 integron integrase (AY260546.3)
intI2
prob_intI2_11
ireA
ireA_20
class 2 integron integrase (AY183453.1)
siderophore receptor - iron-regulated outer
membrane protein (AF320691.1)
iroN
iroN_10
outer membrane siderophore receptor (AF449498.2)
iss
iss_10
increased serum survival (AF042279.1)
katP
hp_katP_611
hp_tir_4051.6_611,
hp_tir_MPEC_611,
hp_tir_O103H2_611,
hp_tir_O111_611,
hp_tir_O157H45_611,
hp_tir_O157H7_611,
hp_tir_NTH19_611
peroxidase and catalase (AB011549.2)
tir
E. coli combined Assay
User Guide 13-11-11-001-V1
translocated intimin receptor (consensus)
40
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APPENDIX 3 – TYPING INFORMATION
Definitions & Explanations
The displayed result will yield following typing information:

Discrimination of the 23 described O-serotypes is mainly determined by the genes wzy
(polymerase) and wzx (flippase). The 47 known H-antigens are encoded by the gene fliC.

The probes immobilized on the current array version can discriminate 23 O-antigens:
O:4, O:6, O:7, O:8, O:9, O:15, O:26, O:52, O:53, O:55, O:79, O:86, O:91, O:101, O:103,
O:104, O:111, O:113, O:114, O:121, O:128, O:157 and O:172

The following flagellar antigens can be identified on the array:
H:01, H:02, H:03, H:04, H:05, H:06, H:07, H:08, H:09, H:10, H:11, H:12, H:14, H:15, H:16,
H:18, H:19, H:20, H:21, H:23, H:24, H:25, H:26, H:27, H:28, H:29, H:30, H:31, H:32, H:33,
H:34, H:37, H:38, H:39, H:40, H:41, H:42, H:43, H:45, H:46, H:46, H:48, H:49, H:51, H:52,
H:53, H:54, H:56

The mix culture control is based on 5 probes located in consensus sequences on the 5’ end
of the fliC genes. These probes were divided in two groups which are correlated with two
groups of fliC genes (fliC-5p_11, fliC-5p_12 and fliC-5p_13, fliC-5p_14, fliC-5p_15). Only one
group should be positive, if not an E. coli mix culture was probed.

The gene fljA encodes for a repressor protein which repressed some of fliC genes. If the fljA
probe positive may two H- serotype probes are positive. In this case the genes flkA and
flmA encodes for the H-serotype. Such strains are described as biphasic (Wang et al. 2003,
Tominaga 2004).

Probes specifying gapA, gad, ihfA, and dnaE that were introduced to confirm the identity of
E. coli and to serve as genus controls.

Probes used to detect the following antimicrobial resistance and virulence genes: see
Appendix 2 -Probe to Target Table
E. coli combined Assay
User Guide 13-11-11-001-V1
41
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