Urine-Based HSV-1&2 PCR Detection Kit - Protocol

3430 Schmon Parkway
Thorold, ON, Canada L2V 4Y6
Phone: 866-667-4362 • (905) 227-8848
Fax: (905) 227-1061
Email: techsupport@norgenbiotek.com
Urine - Based HSV-1&2 PCR Detection Kit
Product Insert
Product # 31700
Herpes Simplex Virus 1 and 2 (HSV-1&2) are both members of the herpes virus family, Herpesviridae.
HSV-1 and HSV-2 have relatively large double-stranded DNA genomes. HSVs are primarily
transmitted by sexual intercourse, direct contact with lesions or perinatally. Most HSV positive cases
are characterised by lesions on the skins and mucous membranes of the mouth and genitals. HSV
infection can be either primary or a recurrence of a previous infection. More than 90% of the primary
HSV infections are asymptomatic. Primary infection with HSV-1 can lead to gingivostomatitis, eczema
herpeticum, keratoconjunctivitis and encephalitis. The primary symptoms of a secondary infection are
skin lesions in the nose, mouth and genital regions. The infection is contagious, mainly during an
epidemic.
Principle of the Test
Norgen’s Urine-Based HSV-1&2 PCR Detection Kit constituents a ready-to-use system for the
isolation, detection and differentiation of HSV-1 and HSV-2 using end-point PCR. The kit first allows for
the isolation of total DNA, including viral DNA, from the urine samples using spin-column
chromatography based on Norgen’s proprietary resin. The viral DNA is isolated free from inhibitors,
and can then be used as the template in a PCR reaction for HSV-1 and HSV-2 detection using the
provided HSV-1&2 Master Mix. The HSV-1&2 Master Mix contain reagents and enzymes for the
specific amplification of a 275bp region of HSV-1 and 350 bp region of HSV-2. In addition, Norgen’s
Urine-Based HSV-1&2 PCR Detection Kit contains a second heterologous amplification system to
identify possible PCR inhibition and/or inadequate isolation. The amplification and detection of either
the HSV-1&2 Isolation Control (IsoC) or the PCR control (PCRC) does not reduce the detection limit of
the analytical HSV-1 or HSV-2 PCR. The kit is designed to allow for the testing of 24 samples.
Kit Components:
Component
Contents
Binding Solution I
20 mL
Proteinase K
0.6 mL
Pronase
0.6 mL
Binding Solution II
3 mL
Wash Solution I
4 mL
Wash Solution II
12 mL
Elution Buffer
3 mL
Mini Filter Spin Columns
24
Collection Tubes
24
Elution tubes (1.7 mL)
24
HSV-1&2 2x PCR Master Mix
0.35 mL
a
HSV-1&2 Isolation Control (IsoC)
b
a
0.4 mL
HSV-1&2 Positive Control (PosC)
0.1 mL
HSV-1&2 Negative Control (NegC)
1.25 mL
Norgen’s DNA Marker
0.1 mL
Product Insert
1
The positive control is a cloned HSV-1 and HSV-2 product
The isolation control is a cloned PCR product
b
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Customer-Supplied Reagents and Equipment
• Disposable powder-free gloves
• Centrifuge with a swinging bucket rotor capable of 2000 x g
• Benchtop microcentrifuge
• Micropipettors
• Sterile pipette tips with filters
• PCR tubes
• 96 – 100% ethanol
• 60°C incubator
• 15 mL tubes
Storage Conditions and Product Stability
All buffers should be kept tightly sealed and stored at room temperature (15-25oC) for up to 1 year
without showing any reduction in performance.
Norgen’s Urine-Based HSV-1&2 PCR Detection Kit contains ready-to-use Proteinase K and Pronase
solutions, which are dissolved in a specially prepared storage buffer. The Proteinase K and the
Pronase are stable for up to 1 year after delivery when stored at room temperature. To prolong the
lifetime of Proteinase K and Pronase, storage at 2–8°C is recommended.
The HSV-1&2 2x PCR Master Mix, the HSV-1&2 Isolation Control (IsoC), the HSV-1&2 Positive
Control (PosC) and the HSV-1&2 Negative Control (NegC) should be kept tightly sealed and stored at
-20oC for up to 1 year without showing any reduction in performance. Repeated thawing and freezing
(> 2 x) should be avoided, as this may reduce the sensitivity. If the reagents are to be used only
intermittently, they should be frozen in aliquots.
General Precautions
The user should exercise the following precautions while using the kit:
• Use sterile pipette tips with filters.
• Store and extract positive material (specimens, controls and amplicons) separately from all
other reagents and add it to the reaction mix in a spatially separated facility.
• Thaw all components thoroughly at room temperature before starting an assay.
• When thawed, mix the components and centrifuge briefly.
• Work quickly on ice.
Quality Control
In accordance with Norgen’s ISO 9001 and ISO 13485-certified Quality Management System, each lot
of Norgen’s Urine-Based HSV-1&2 PCR Detection Kit, the HSV-1&2 2x PCR Master Mix, the HSV-1&2
Isolation Control (IsoC), the HSV-1&2 Negative Control (NegC) and the HSV-1&2 Positive Control
(PosC) are tested against predetermined specifications to ensure consistent product quality.
Product Use Limitations
Norgen’s Urine-based HSV-1&2 PCR Detection Kit is designed for research purposes only. It is not
intended for human or diagnostic use.
Product Warranty and Satisfaction Guarantee
NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner
described in our product manual. The customer must determine the suitability of the product for its
particular use.
Safety Information
Ensure that a suitable lab coat, disposable gloves and protective goggles are worn when working with
chemicals. For more information, please consult the appropriate Material Safety Data Sheets
(MSDSs). These are available as convenient PDF files online at www.norgenbiotek.com.
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CAUTION: DO NOT add bleach or acidic solutions directly to the sample-preparation waste.
The Binding Solution I, Binding Solution II, Wash Solution I and Wash Solution II contain
guanidine hydrochloride, and should be handled with care. Guanidine hydrochloride forms highly
reactive compounds when combined with bleach, thus care must be taken to properly dispose of any
of these solutions.
If liquid containing these buffers is spilled, clean with suitable laboratory detergent and water. If the
spilled liquid contains potentially infectious agents, clean the affected area first with laboratory
detergent and water, and then with 1% (v/v) sodium hypochlorite.
1. Protocol
A. Specimen Collection, Storage and Transport
Precaution: All samples have to be treated as potentially infectious material.
1. Specimen Collection and Sample Storage
• Midstream urine samples should be collected, as the first flow of urine has been shown to
have a higher rate of contamination (Morimoto et al., 2003).
• It is highly recommended that urine samples be collected using Norgen’s Urine Collection and
Preservation Tubes (Cat# 18111). The urine samples can be stored for at least one year at
room temperature when collected directly using Norgen’s Urine Collection and Preservation
Tubes.
• Alternatively, urine samples collected using any other collection and preservation systems or
reagents are also compatible with this kit.
2. Sample Transport
• Sample material should be transported in a shatterproof, leak-proof transport container as a
matter of principle. Thus, a potential danger of infection due to a leakage of sample can be
avoided.
• The samples should be transported following the local and national instructions for the
transport of pathogen material.
B. Isolation of DNA from Urine
Notes:
• Do not spin down or filter the urine sample before proceeding with the isolation, as this could
negatively affect the isolation of viral DNA.
• Ensure that all solutions are at room temperature prior to use, and that no precipitates have
formed. If necessary, warm the solutions and mix well until the solutions become clear again.
• Always vortex both the Proteinase K and the Pronase before use.
• Preheat an incubator or heating block to 60°C.
• Prepare a working concentration of Binding Solution II and Wash Solution I by adding the
proper volume of 96-100% ethanol (provided by the user) indicated in Table 1 below to the
supplied bottle containing the concentrated Binding Solution II and Wash Solution I. The label
on the bottle has a box that may be checked to indicate that the ethanol has been added.
• Elevated levels of bilirubin (≥15 mg/dl) and lipids (≥800 mg/dl) and haemolytic samples do not
influence the system
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Table 1. Volume of Ethanol to be added to Binding Buffer II and Wash Buffer I
Volume Provided
Ethanol (96-100%) Volume to Add
Final Volume
Binding Solution II
3 mL
7 mL
10 mL
Wash Solution I
4 mL
11 mL
15 mL
•
An HSV-1&2 Isolation Control (IsoC) is supplied. This allows the user to control the DNA
isolation procedure. For this assay, add the HSV-1&2 Isolation Control (IsoC) to the lysate
during the isolation procedure.
o The HSV-1&2 Isolation Control (IsoC) must not be added to the sample material
directly
o Do not freeze and thaw the HSV-1&2 Isolation Control (IsoC) more than 2 times.
o The HSV-1&2 Isolation Control (IsoC) must be kept on ice at all times during the
isolation procedure.
1.
Obtain a 5 mL midstream urine sample. Add 700 µL of Binding Solution I to the urine sample
and mix well by inversion. (Note: Binding Solution I contains resin and must be mixed well
before every pipeting).
2.
Centrifuge for 5 minutes at 2,000 x g. Discard the supernatant.
3.
Add 20 µL of both Proteinase K and Pronase to the precipitated slurry pellet resulting from 5 mL
of the urine sample. Vortex for 10 seconds.
4.
Incubate the mixture at 60°°C for 20 minutes.
5.
After the 20 minute incubation, add 260µL Binding Solution II,
6.
Add 15 µL HSV-1&2 Isolation Control (IsoC) to the lysate, mix well by pipeting and then transfer
the entire contents into a Mini Filter Spin column (provided).
7.
Centrifuge for 2 minutes at 6,700 x g, and discard the flow-through.
8.
Apply 450 µL of Wash Solution I to the column and centrifuge for 1 minute. Discard the
flowthrough and reassemble the spin column with its collection tube.
9.
Apply 450 µL of Wash Solution II to the column and centrifuge for 1 minute. Discard the flowthrough and reassemble the spin column with its collection tube.
10. Spin the column for 1 minute in order to thoroughly dry the resin. Discard the collection tube.
11. Transfer the spin column to a fresh 1.7 mL Elution tube. Apply 100 µL of Elution Buffer to the
column and centrifuge for 2 minutes at 200 x g (~2,000 RPM), followed by 1 minute at 14,000 x
g (~14,000 RPM).
C. HSV-1&2 PCR Assay Preparation
Notes:
• It is recommended that 10 µL of the DNA elution be used as the PCR sample input volume
• Sample volume can be varied between 2 µL – 10 µL of the DNA elution. PCR grade water
should be added to make up the final volume of the PCR reaction to 20 µL.
• Using a lower volume from the sample than recommended may affect the sensitivity of the
HSV-1&2 Limit of Detection.
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•
•
•
1.
2.
3.
4.
HSV-1&2 Negative Control (NegC) and HSV-1&2 Positive Control (PosC) must be included
during every run.
The HSV-1&2 Negative Control (NegC) and HSV-1&2 Positive Control (PosC) provided are
sufficient for eight PCR runs.
Before each use, all reagents need to be thawed completely, mixed (by repeated up and
down pipetting or quick vortexing), and centrifuged briefly.
Prepare PCR reactions as outlined in Table 2 below. For each sample to be run, pipette 10
µL of the eluted DNA and 10 µL of the Master Mix into a PCR tube. Each PCR reaction will
have a final volume of 20 µL.
HSV-1&2 Negative Control (NegC) and HSV-1&2 Positive Control (PosC) must be included in
every run. Pipette 10 µL of HSV-1&2 Negative Control (NegC) into a PCR tube and add 10
µL of Master Mix. Pipette 10 µL of HSV-1&2 Positive Control (PosC) into a PCR tube and add
10 µL of Master Mix.
Program the PCR machine according to the program shown in Table 3 below.
Run PCR.
Table 2. PCR Assay Preparation
Preparation of PCR assay
Volume Per PCR Reaction
HSV-1&2 2X PCR Master Mix
10 µL
Sample (Eluted DNA)
10 µL
HSV-1&2 Positive Control (PosC)
10 µL
10 µL
----------
10 µL
----------
HSV-1&2 Negative Control (NegC)
----------
----------
10 µL
Total Volume
20 µL
20 µL
20 µL
Table 3. HSV-1&2 PCR Assay Program
PCR Cycle
Cycle 1
Cycle 2 (40x)
Cycle 3
Cycle 4
Step
Temperature
Duration
Step 1
o
95 C
3 min
Step 1
o
94 C
15 sec
Step 2
o
60 C
30 sec
Step 3
o
72 C
45 sec
Step 1
o
72 C
5 min
Step 1
o
4C
∞
D. HSV-1&2 PCR Assay Interpretation
•
•
•
For the analysis of the PCR data, the entire 20 µL PCR reaction should be loaded on a 1X
TAE, 2% Agarose DNA gel along with 10 µL of Norgen’s DNA Marker (provided).
The PCR products should be resolved on the 1X TAE, 2% Agarose gel at 150V for 30 minutes
Figure 1 and Table 4 explain how to interpret the PCR assay results
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M
A
B
C
D
E
F G NegC M 1
2
3
4
5
6
7 NegC M
2000
1500
1000
750
Isolation Control
(IsoC)
500
300
HSV2 Target
HSV1Target
150
PCR Control
(PCRC)
50
Figure 1: A representative 1X TAE, 1.7% agarose gel showing the amplification of HSV-1 and HSV-2 at
different concentrations (Target). The size of the HSV-1 target amplicon corresponds to the 275bp band
represented by the provided DNA Marker (M).The size of the HSV-2 target amplicon corresponds to the
350bp band represented by the provided DNA Marker (M). The size of the HSV-1&2 Isolation Control (IsoC)
corresponds to the 500bp band represented by the provided DNA Marker (M). The HSV-1&2 2X PCR
Master Mix contains a HSV-1&2 PCR Control (PCRC). The HSV-1&2 PCRC Controls for PCR inhibition.
The size of the HSV-1&2 PCRC corresponds to the 150bp band represented by the provided DNA Marker
(M). Lanes A-G represents samples spiked with different HSV-1 concentrations isolated from 5mL urine
samples (interpreted as positive results). Lanes 1-7 represents samples spiked with different HSV-2
concentrations isolated from 5mL urine samples (interpreted as positive results).The HSV-1 and HSV-2
spiked in urine samples is a cloned PCR product.
Table 4. Interpretation of PCR Assay Results
Input Type
HSV-1&2 IsoC
Band (500 bp)
HSV-1 (275
bp) or HSV-2
(350 bp)
Target Band
HSV-1&2 PCRC
Band (150 bp)
Interpretation
Positive Control
X
X
X
Valid
X
Valid
X
Positive
X
Negative
X
Positive
Negative Control
Sample
X
Sample
X
Sample
Sample
Sample
X
X
X
X
Positive
X
Positive
** For results obtained that are not covered in Table 4 above, please refer to the Troubleshooting
Section.
E. Specificity
The specificity of Norgen’s Urine-Based HSV-1&2 PCR Detection Kit is first and foremost ensured by
the selection of the HSV-1 and HSV-2-specific primers, as well as the selection of stringent reaction
conditions. The primers were checked for possible homologies in GenBank published sequences by
sequence comparison analyses. Furthermore, the specificity of the HSV-1 and HSV-2-specific primers
were tested against most of the known sexually-transmitted pathogens.
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F. Linear Range
•
•
•
•
The linear range (analytical measurement) of Norgen’s Urine-Based HSV-1&2 PCR Detection
Kit was determined by analyzing a dilution series of HSV-1 and HSV-2 quantitative standard
ranging from 8.46 x 109 VP/µl to 1 x 10-1 IU/µl.
Each dilution has been tested in replicates (n = 4) using Norgen’s Urine-Based HSV-1&2 PCR
Detection Kit on 1X TAE, 1.7% Agarose gels.
The linear range of Norgen’s Urine-Based HSV-1&2 PCR Detection Kit has been determined
to cover concentrations from 0.2 VP/µl to at least 8 x 106 VP/µl
Under the conditions of Norgen’s Urine DNA Isolation procedure, Norgen’s Urine-Based HSV1&2 PCR detection Kit covers a linear range from 200VP/mL urine to at least 8 x 109 VP/mL
urine.
G. Frequently Asked Questions
1. How many samples should be included per PCR run?
• Norgen’s Urine-Based HSV-1&2 PCR Detection Kit is designed to test 24 samples. For every 6
samples, a Negative Control and a Positive Control must be included. It is preferable to pool and test 6
samples at a time. If not, the provided Negative Control and Positive Control are enough to run 3
samples at a time.
2. How can I interpret my results for a sample if neither the HSV-1&2 PCR control nor the HSV-1&2
Isolation Control amplifies?
• If neither the HSV-1&2 PCR control nor the HSV-1&2 Isolation Control (IsoC) amplifies, the sample
must be re-tested. If the positive control showed amplification, then the problem occurred during the
isolation, where as if the Positive control did not amplify the problem has occurred during the setup of
the PCR assay reaction.
3. How should it be interpreted if only the HSV-1&2 PCR control showed amplification but neither
the HSV-1 or HSV-2 target nor the HSV-1&2 Isolation Control (IsoC) amplified for a sample?
• This indicates a poor isolation. The isolation procedure must be repeated.
4. How should it be interpreted if only the HSV-1&2 Isolation Control (IsoC) was amplified in a
sample?
• The sample tested can be considered as HSV-1 or HSV-2 negative.
5. How should it be interpreted if only the HSV-1 or HSV-2 target and the HSV-1&2 PCR control were
amplified in a sample?
• The sample tested can be considered as HSV-1 or HSV-2 positive.
6. How should it be interpreted if only the HSV-1 target was amplified in a sample?
• The sample tested can be considered positive. At high HSV-1 viral load, the HSV-1 amplicon will be
predominant and the HSV-1&2 PCR control as well as the HSV-1&2 Isolation control may not amplify.
7. How should it be interpreted if only the HSV-2 target was amplified in a sample?
• The sample tested can be considered positive. At high HSV-2 viral load, the HSV-2 amplicon will be
predominant and the HSV-1&2 PCR control as well as the HSV-1&2 Isolation control may not amplify.
8. How should it be interpreted if only the HSV-1&2 PCR control and the HSV-1&2 Isolation Control
(IsoC) showed amplification?
• The sample tested can be considered negative
9. Can I process a different urine volume?
• The reagents provided with the isolation kit are only sufficient to process 24 urine samples of 5mL
each.
9. What If I added more or less of the specified reagents’ volume during DNA isolation?
• Adding less volume may reduce your DNA yields. Adding more may not affect the DNA yields EXCEPT
if more Elution Buffer was added. Eluting DNA in higher volumes of Elution Buffer will result in diluting
your DNA.
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10. What If my incubation temperature varied from the specified 60°°C?
• The incubation temperature can be in the range of 55°C - 65°C. At other temperatures the activity of
both the Proteinase K and the Pronase will be reduced. This will result in a reduction in your DNA
yields.
11. What If my incubation varied from the 20 minutes specified in the product manual?
• Less than 20 minutes will result in lower DNA yields. More than 20 minutes may not affect your DNA
yields.
12. What If I forgot to do a dry spin after my second wash?
• Your DNA elution will be contaminated with the Wash Solution. This may dilute the DNA yield in your
elution and it may interfere with your down stream applications.
13. What If I forgot to add the HSV-1&2 Isolation control during the Isolation?
• The Isolation must be repeated.
Related Products
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Urine Bacteria RNA Purification Kit
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Urine-Based HSV-1 PCR Detection Kit
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Urine-Based HSV-2 PCR Detection Kit
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Technical Assistance
NORGEN’s Technical Service Department is staffed by experienced scientists with extensive practical and
theoretical expertise in sample and assay technologies and the use of NORGEN products. If you have any
questions or experience any difficulties regarding Norgen’s Urine-based HSV-2 PCR Detection Kit or
NORGEN products in general, please do not hesitate to contact us.
NORGEN customers are a valuable source of information regarding advanced or specialized uses of our
products. This information is helpful to other scientists as well as to the researchers at NORGEN. We
therefore encourage you to contact us if you have any suggestions about product performance or new
applications and techniques.
For technical assistance and more information, please contact our Technical Support Team between the
hours of 8:30 and 5:30 (Eastern Standard Time) at (905) 227-8848 or Toll Free at 1-866-667-4362 or call
one of the NORGEN local distributors (www.norgenbiotek.com) or through email at
techsupport@norgenbiotek.com.
References
Morimoto, M., Yanai, H., Chiba, H., Matsuno, K. and Shukuya, K. (2003). Importance of midstream cleancatch technique for urinalysis, reconfirmed by urinary flow cytometry. Clin Chim Acta. 333, 101-102.
3430 Schmon Parkway, Thorold, ON Canada L2V 4Y6
Phone: (905) 227-8848
Fax: (905) 227-1061
Toll Free in North America: 1-866-667-4362
©2010 Norgen Biotek Corp.
PI31700-5
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