M1 - global Influenza A
&
N1- specific for Swine H1N1
Influenza – Human Pandemic
Strain
Swine H1N1 Influenza
Human Pandemic
Strain
PCRmax Ltd qPCR test
TM
150 tests
For general laboratory and research use only
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
1
Influenza, commonly known as the flu, is an infectious disease of birds and mammals
caused by an RNA virus of the family Orthomyxoviridae (the influenza viruses). In people,
common symptoms of influenza are fever, sore throat, muscle pains, severe headache,
coughing, and weakness and fatigue. In more serious cases, influenza causes
pneumonia, which can be fatal, particularly in young children and the elderly. Although the
common cold is sometimes confused with influenza, it is a much less severe disease and
caused by a different virus. Similarly, gastroenteritis is sometimes called "stomach flu" or
"24-hour flu", but is unrelated to influenza.
Typically, influenza is transmitted from infected mammals through the air by coughs or
sneezes creating aerosols containing the virus, and from infected birds through their
droppings. Influenza can also be transmitted by saliva, nasal secretions, feces and blood.
Infections either occur through direct contact with these bodily fluids, or by contact with
contaminated surfaces. Flu viruses can remain infectious for over 30 days at 0°C (32°F)
and about one week at human body temperature, although they are rapidly inactivated by
disinfectants and detergents.
Flu spreads around the world in seasonal epidemics, killing millions of people in pandemic
years and hundreds of thousands in non-pandemic years. Three influenza pandemics
occurred in the 20th century – each following a major genetic change in the virus – and
killed tens of millions of people. Often, these pandemics result from the spread of a flu
virus between animal species. Since it first killed humans in Asia in the 1990s a deadly
avian strain of H5N1 has posed the greatest influenza pandemic threat. However, this virus
has not yet mutated to spread easily between people.
Vaccinations against influenza are most common in high-risk humans in industrialised
countries and farmed poultry. The most common human vaccine is the trivalent flu vaccine
that contains purified and inactivated material from three viral strains. Typically this vaccine
includes material from two influenza A virus subtypes and one influenza B virus strain. A
vaccine formulated for one year may be ineffective in the following year, since the
Influenza A Virus changes every year and different strains become dominant. Antiviral
drugs can be used to treat influenza, with neuraminidase inhibitors being particularly
effective.
Introduction to Swine H1N1 Influenza Human
Pandemic Strain
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
2
Specificity
The kit has 2 primer and probe sets. The M1 primers and probe detect ALL influenza A
isolates including those originally isolated from both human and swine. The primers have
100% homology to all pandemic H1N1 sequences.
The N1 primer and probe set detects ONLY the pandemic swine flu H1N1. The primers
and probe have 100% homology to all pandemic strains. The primers will not detect N1
from any seasonal endemic human or swine N1 strains.
The PCRmax qPCR Kit for Swine H1N1 Influenza Human Pandemic Strain (H1N1-swine)
genomes is designed for the in vitro quantification of H1N1-swine genomes. The kit is
designed to have the broadest detection profile possible whilst remaining specific to the
H1N1-swine genome.
The primers and probe sequences in this kit have 100% homology with a broad range of
H1N1-swine sequences based on a comprehensive bioinformatics analysis.
If you require further information, or have a specific question about the detection profile of
this kit then please send an e.mail to help@pcrmax.com and our bioinformatics team will
answer your question.
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
3
Kit Contents
• M1 global influenza A primer/probe mix (150 reactions BROWN)
FAM labelled
• N1 swine H1N1 influenza specific primer/probe mix (150 reactions BROWN)
FAM labelled
• M1 global influenza A positive control template (for Standard curve RED)
• N1 swine H1N1 influenza specific positive control template (for Standard curve
RED)
• Internal extraction control primer/probe mix (150 reactions BROWN)
VIC labelled as standard
• Internal extraction control RNA (150 reactions BLUE)
• Endogenous control primer/probe mix (150 reactions BROWN)
FAM labelled
• M1 and N1/Internal extraction control/endogenous control RT primer mix (150
reactions GREEN)
Required for two step protocol only
• RNAse/DNAse free water (WHITE)
for resuspension of primer/probe mixes and internal extraction control RNA
• Template preparation buffer (YELLOW)
for resuspension of positive control templates and standard curve preparation
Reagents and equipment to be supplied by the user
Real-Time PCR Instrument
RNA extraction kit
This kit is designed to work well with all processes that yield high quality RNA with
minimal PCR inhibitors.
Lyophlised 2x RT-qPCR MasterMix
This kit is designed to be compatible with all commercially available OneStep
Mastermixes that run with standard cycling conditions.
Pipettors and Tips
Vortex and centrifuge
Thin walled 1.5 ml PCR reaction tubes
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
4
Kit storage and stability
This kit is stable at room temperature but should be stored at -20ºC on arrival. Once the
lyophilised components have been resuspended they should not be exposed to
temperatures above -20ºC for longer than 30 minutes and unnecessary repeated
freeze/thawing should be avoided. The kit is stable for six months from the date of
resuspension under these circumstances.
If a standard curve dilution series is prepared this can be stored frozen for an extended
period. If you see any degradation in this serial dilution a fresh standard curve can be
prepared from the positive control.
PCRmax does not recommend using the kit after the expiry date stated on the pack.
Suitable sample material
All kinds of sample material suited for PCR amplification can be used. Please ensure the
samples are suitable in terms of purity, concentration, and RNA/DNA integrity (An internal
PCR control is supplied to test for non specific PCR inhibitors). Always run at least one
negative control with the samples. To prepare a negative-control, replace the template
RNA sample with RNAse/DNAse free water.
Dynamic range of test
Under optimal PCR conditions PCRmax H1N1-swine detection kits have very high priming
efficiencies of >95% and can detect less than 100 copies of target template.
Notices and disclaimers
This product is developed, designed and sold for research purposes only. It is not intended for human diagnostic or drug
purposes or to be administered to humans unless clearly expressed for that purpose by the Food and Drug Administration in
the USA or the appropriate regulatory authorities in the country of use. During the warranty period PCRmax detection kits
allow precise and reproducible data recovery combined with excellent sensitivity. For data obtained by violation to the
general GLP guidelines and the manufacturer’s recommendations the right to claim under guarantee is expired. PCR is a
proprietary technology covered by several US and foreign patents. These patents are owned by Roche Molecular Systems
Inc. and have been sub-licensed by PE Corporation in certain fields. Depending on your specific application you may need a
license from Roche or PE to practice PCR. Additional information on purchasing licenses to practice the PCR process may
be obtained by contacting the Director of Licensing at Roche Molecular Systems, 1145 Atlantic Avenue, Alameda, CA 94501
or Applied Biosystems business group of the Applera Corporation, 850 Lincoln Centre Drive, Foster City, CA 94404. In
addition, the 5' nuclease assay and other homogeneous amplification methods used in connection with the PCR process
may be covered by U.S. Patents 5,210,015 and 5,487,972, owned by Roche Molecular Systems, Inc, and by U.S. Patent
5,538,848, owned by The Perkin-Elmer Corporation.
Trademarks
PCRmax™ is a trademark of PCRmax Ltd.
The PCR process is covered by US Patents 4,683,195, and 4,683,202 and foreign equivalents owned by Hoffmann-La
Roche AG. BI, ABI PRISM® GeneAmp® and MicroAmp® are registered trademarks of the Applera Genomics (Applied
Biosystems Corporation). BIOMEK® is a registered trademark of Beckman Instruments, Inc.; iCycler™ is a registered
trademark of Bio-Rad Laboratories, Rotor-Gene is a trademark of Corbett Research. LightCycler™ is a registered trademark
of the Idaho Technology Inc. GeneAmp®, TaqMan® and AmpliTaqGold® are registered trademarks of Roche Molecular
Systems, Inc., The purchase of the PCRmax reagents cannot be construed as an authorization or implicit license to practice
PCR under any patents held by Hoffmann-LaRoche Inc.
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
5
Principles of the test
The kit contains two Influenza A primer and probe sets. The M1 primer and probe set are
designed to detect all influenza A sequences regardless of the species of origin. Samples
that test positive with the M1 primer set contain an influenza A infection of unknown
serotype. The N1 primer and probe set is specific to the swine H1N1 pandemic strain.
Samples that test positive with the N1 primer contain the pandemic swine H1N1 Influenza.
Samples that test positive for M1 and negative for N1 are indicative of a naturally
circulating influenza A infection which is not swine flu.
Real-time PCR
A H1N1-swine specific primer and probe mix is provided and this can be detected through
the FAM channel.
The primer and probe mix provided exploits the so-called TaqMan® principle. During PCR
amplification, forward and reverse primers hybridize to the H1N1-swine cDNA. A
fluorogenic probe is included in the same reaction mixture which consists of a DNA probe
labeled with a 5`-dye and a 3`-quencher. During PCR amplification, the probe is cleaved
and the reporter dye and quencher are separated. The resulting increase in fluorescence
can be detected on a range of real-time PCR platforms.
Positive control
For copy number determination and as a positive control for the PCR set up, the kit
contains a positive control template. This can be used to generate a standard curve of
H1N1-swine copy number / Cq value. Alternatively the positive control can be used at a
single dilution where full quantitative analysis of the samples is not required. Each time the
kit is used, at least one positive control reaction must be included in the run. A positive
result indicates that the primers and probes for detecting the target H1N1-swine gene
worked properly in that particular experimental scenario. If a negative result is obtained
the test results are invalid and must be repeated. Care should be taken to ensure that the
positive control does not contaminate any other kit component which would lead to falsepositive results. This can be achieved by handling this component in a Post PCR
environment. Care should also be taken to avoid cross-contamination of other samples
when adding the positive control to the run. This can be avoided by sealing all other
samples and negative controls before pipetting the positive control into the positive control
well.
Negative control
To validate any positive findings a negative control reaction should be included every time
the kit is used. For this reaction the RNAse/DNAse free water should be used instead of
template. A negative result indicates that the reagents have not become contaminated
while setting up the run.
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
6
Internal RNA extraction control
When performing RNA extraction, it is often advantageous to have an exogenous source
of RNA template that is spiked into the lysis buffer. This control RNA is then co-purified
with the sample RNA and can be detected as a positive control for the extraction process.
Successful co-purification and real-time PCR for the control RNA also indicates that PCR
inhibitors are not present at a high concentration.
A separate RT primer mix and a real-time PCR primer/probe mix are supplied with this kit
to detect the exogenous RNA using real-time PCR. The PCR primers are present at PCR
limiting concentrations which allows multiplexing with the target sequence primers.
Amplification of the control cDNA does not interfere with detection of the H1N1-swine
target cDNA even when present at low copy number. The Internal control is detected
through the VIC channel and gives a Cq value of 28+/-3 depending on the level of sample
dilution.
Endogenous control
To confirm extraction of a valid biological template, a primer and probe mix is included to
detect an endogenous gene. Detection of the endogenous control is through the FAM
channel and it is NOT therefore possible to perform a multiplex with the H1N1-swine
primers. A poor endogenous control signal may indicate that the sample did not contain
sufficient biological material.
Carry-over prevention using UNG (unsuitable for onestep procedure and optional for
two step procedure)
Carry over contamination between PCR reactions can be prevented by including uracil-Nglycosylase (UNG) in the reaction mix. Some commercial mastermix preparations contain
UNG or alternatively it can be added as a separate component. UNG can only prevent
carry over from PCR reactions that include deoxyuridine triphosphate (dUTP) in the
original PCR reaction. PCRmax recommend the application of 0.2U UNG per assay with
a 15 minute incubation step at 37°C prior to amplification. The heat-labile UNG is then
inactivated during the Taq polymerase activation step.
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
7
Component - resuspend in water
Volume
M1 primer/probe mix (BROWN)
165 µl
Internal extraction control RNA (BLUE)
600 µl
Internal extraction control primer/probe mix (BROWN)
Endogenous control primer/probe mix (BROWN)
Pre-PCR pack
RT primer mix (GREEN)
Pre-PCR heat-sealed foil
165 µl
165 µl
165 µl
Reconstitution Protocol
To minimize the risk of contamination with foreign DNA, we recommend that all pipetting
be performed in a PCR clean environment. Ideally this would be a designated PCR lab or
PCR cabinet. Filter tips are recommended for all pipetting steps.
1. Pulse-spin each tube in a centrifuge before opening.
This will ensure lyophilised primer and probe mix is in the base of the tube and is not
spilt upon opening the tube.
2. Reconstitute the kit components in the RNAse/DNAse free water supplied,
according to the table below:
To ensure complete resuspension, vortex each tube thoroughly.
* This component contains high copy number template and is a VERY significant contamination
risk. It must be opened and handled in a separate laboratory environment, away from the other
components.
RNA extraction
The internal extraction control RNA can be added either to the RNA lysis/extraction buffer
or to the RNA sample once it has been resuspended in lysis buffer.
DO NOT add the internal extraction control RNA directly to the unprocessed biological
sample as this will lead to degradation and a loss in signal.
1. Add 4µl of the Internal extraction control RNA (BLUE) to each sample in RNA
lysis/extraction buffer per sample.
2. Complete RNA extraction according to the manufacturers protocols.
N1 primer/probe mix (BROWN)
165 µl
3. Reconstitute the positive control templates in the template preparation buffer
supplied, according to the table below:
To ensure complete resuspension, vortex each tube thoroughly.
500 µl
Post-PCR heat-sealed foil
Component - resuspend in template preparation buffer
Volume
M1 Positive Control Template (RED) *
N1 Positive Control Template (RED) *
500 µl
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
8
One Step qRT-PCR detection protocol
A one step approach combining the reverse transcription and amplification in a single
closed tube is the preferred method.
For optimum performance and sensitivity.
All pipetting steps and experimental plate set up should be performed on ice. After the
plate is poured proceed immediately to the One Step amplification protocol. Prolonged
incubation of reaction mixes at room temperature can lead to PCR artifacts that reduce
the sensitivity of detection.
Component
Volume
Lyophilised OneStep 2x RT-qPCR MasterMix
1 µl
M1 or N1 primer/probe mix (BROWN)
Final Volume
1 µl
15 µl
10 µl
Internal extraction control primer/probe mix (BROWN)
RNAse/DNAse free water (WHITE)
3 µl
2. For each RNA sample prepare an endogenous control reaction according to
the table below (optional):
This control reaction will provide crucial information regarding the quality of the
biological sample.
Component
Volume
Lyophilised OneStep 2x RT-qPCR MasterMix
1 µl
Endogenous control primer/probe mix (BROWN)
Final Volume
15 µl
10 µl
RNAse/DNAse free water (WHITE)
4 µl
1. For each RNA sample prepare a reaction mix according to the table below:
Include sufficient reactions for positive and negative controls.
3. Pipette 15µl of these mixes into each well according to your real-time PCR
experimental plate set up.
4. Pipette 5µl of RNA template into each well, according to your experimental
plate set up.
For negative control wells use 5µl of RNAse/DNAse free water. The final volume in
each well is 20µl.
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
9
Step
Reverse Transcription
Enzyme activation
Denaturation
DATA COLLECTION *
Time
Temp
10 mins
2 mins
10 secs
60 secs
55 oC
95 oC
95 oC
60 oC
Cycling x 50
One Step Amplification Protocol
Amplification conditions using Lyophilised OneStep 2x RT-qPCR MasterMix.
* Fluorogenic data should be collected during this step through the FAM and VIC channels
Standard Curve
Copy Number
Tube 1 Positive control (RED)
2 x 105 per µl
Tube 2
Tube 3
Tube 4
Tube 5
Tube 6
2 x 104 per µl
2 x 103 per µl
2 x 102 per µl
20 per µl
2 per µl
6. Preparation of standard curve dilution series.
1) Pipette 90µl of template preparation buffer into 5 tubes and label 2-6
2) Pipette 10µl of Positive Control Template (RED) into tube 2
3) Vortex thoroughly
4) Change pipette tip and pipette 10 µl from tube 2 into tube 3
5) Vortex thoroughly
Repeat steps 4 and 5 to complete the dilution series
7. Pipette 5µl of standard template into each well for the standard curve according
to your plate set-up
The final volume in each well is 20µl.
Component
Volume
Lyophilised OneStep 2x RT-qPCR MasterMix
1 µl
M1 or N1 primer/probe mix (BROWN)
Final Volume
15 µl
10 µl
RNAse/DNAse free water (WHITE)
4 µl
5. If a standard curve is included for quantitative analysis prepare a reaction mix
according to the table below:
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
10
Interpretation of Results
≤ 30
Positive
control
Negative
control
Internal
control
(VIC)
Target
(FAM)
Interpretation
> 30
+ / -
+ / -
-
+ / -
+
-
-
-
> 35
POSITIVE QUANTITATIVE RESULT
calculate copy number
+ / -
+
+ / -++++
≤ 35
EXPERIMENT FAILED
due to test contamination
*
POSITIVE QUANTITATIVE RESULT
calculate copy number
> 30
POSITIVE QUALITATIVE RESULT
do not report copy number as this
may be due to poor sample extraction
-++
-
NEGATIVE RESULT
--+
-
SAMPLE PREPARATION FAILED
+ / -
+ / -
-
+ / -
EXPERIMENT FAILED
*Where the test sample is positive and the negative control is positive with a Cq > 35, the
sample must be reinterpreted based on the relative signal strength of the two results:
Positive control template (RED) is expected to amplify between Cq 16 and 23. Failure to
satisfy this quality control criterion is a strong indication that the experiment has been
compromised.
Sample
Negative
control
∆Cq$>$5
SAMPLE$POSITIVE
Sample
Negative
control
∆Cq$<$5
INCONCLUSIVE
If the sample amplifies > 5 Cq earlier than
the negative control then the sample
should be reinterpreted (via the table
above) with the negative control verified
as negative.
If the sample amplifies < 5 Cq earlier than
the negative control then the positive
sample result is invalidated and the result
should be determined inconclusive due to
test contamination. The test for this
sample should be repeated.
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
11
Internal PCR control
The CT value obtained with the internal control will vary significantly depending on the
extraction efficiency, the quantity of RNA added to the RT and PCR reaction and the
individual machine settings. CT values of 28±3 are within the normal range. When
amplifying a H1N1-swine sample with a high genome copy number, the internal extraction
control may not produce an amplification plot. This does not invalidate the test and should
be interpreted as a positive experimental result.
Endogenous control
The signal obtained from the endogenous control primer and probe set will vary according
to the amount of biological material present in a given sample. An early signal indicates
the presence of a good yield of biological material. A late signal suggests that little
biological material is present in the sample.
Quantification of Swine H1N1 Influenza Human Pandemic Strain genomes
Advanced kit handbook HB10.05.09
Published Date: 26/04/2016
12
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