PCRmax Bird Flu Instruction Manual

Hemagglutinin H5
&
Neuramidase N1

Influenza A virus
subtype H5N1 (avian
influenza)

PCRmax Ltd qPCR test

TM

150 tests

For general laboratory and research use only

Quantification of Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

1

Influenza A virus subtype H5N1, also known as A(H5N1) or H5N1, is a subtype of the
Influenza A virus that is capable of causing illness in many animal species, including
humans.[1] A bird-adapted strain of H5N1, called HPAI A(H5N1) for "highly pathogenic
avian influenza virus of type A of subtype H5N1", is the causative agent of H5N1 flu,
commonly known as "avian influenza" or simply "bird flu" and is endemic in many bird
populations, especially in Southeast Asia. One strain of HPAI A (H5N1) of Asian lineage is
spreading globally. It is epizootic (an epidemic in non-humans) and panzootic (a disease
affecting animals of many species, especially over a wide area), killing tens of millions of
birds and spurring the culling of hundreds of millions of other birds in an attempt to control
its spread. Most references in the media to "bird flu" and to H5N1 are about this specific
strain.[2]

HPAI A(H5N1) is an avian disease, and there is no evidence of efficient human-to-human
transmission or of airborne transmission of HPAI A (H5N1) to humans. In almost all cases,
those infected with H5N1 have had extensive physical contact with infected birds.
However, around 50% of humans known to have been infected with the current Asian
strain of HPAI A(H5N1) have died from H5N1 flu, and H5N1 has the potential to mutate or
re-assort into a strain capable of efficient human-to-human transmission. On September
29, 2005, David Nabarro, the newly-appointed Senior United Nations System Coordinator
for Avian and Human Influenza, warned the world that an outbreak of avian influenza could
kill anywhere between 5 million and 150 million people.[3] Experts have identified key
events (creating new clades, infecting new species, spreading to new areas) marking the
progression of an avian flu virus towards becoming pandemic, and many of those key
events have occurred more rapidly than expected.

References
1 International Committee on Taxonomy of Viruses (2002). 46.0.1. Influenzavirus A.
Retrieved on 2006-04-17.
2 a b Li KS, Guan Y, Wang J, Smith GJ, Xu KM, Duan L, Rahardjo AP, Puthavathana P,
Buranathai C, Nguyen TD, Estoepangestie AT, Chaisingh A, Auewarakul P, Long HT,
Hanh NT, Webby RJ, Poon LL, Chen H, Shortridge KF, Yuen KY, Webster RG, Peiris JS.
(2004). "Genesis of a highly pathogenic and potentially pandemic H5N1 influenza virus in
eastern Asia". Nature 430 (6996): 209-213. PubMedDOI:10.1038/nature02746.
3 United Nations. "Press Conference by UN System Senior Coordinator for Avian, Human
Influenza", UN News and Media Division, Department of Public Information, New York,
2005-09-29. Retrieved on 2006-04-17.

Introduction to Influenza A virus subtype H5N1 (avian
influenza)

Quantification of Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

2

Specificity

The primers have 100% homology with all avian H5 and N1 isolates globally that have
been entered into the influenza sequence database in the NCBI database since 2001.
The primers have low sequence homology to other influenza subtypes. The
quantification of both subtyping genes ensures accurate determination of the H5N1
genotype in a single experiment.

The PCRmax qPCR Kit for Influenza A virus subtype H5N1 (avian influenza) (H5N1)
genomes is designed for the in vitro quantification of H5N1 genomes. The kit is designed
to have the broadest detection profile possible whilst remaining specific to the H5N1
genome.
The primers and probe sequences in this kit have 100% homology with a broad range of
H5N1 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 [e-mail] and our bioinformatics team will
answer your question.

Quantification of Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

3

Kit Contents

• Hemagglutinin H5 primer/probe mix (150 reactions BROWN)

FAM labelled

• Neuramidase N1 primer/probe mix (150 reactions BROWN)

FAM labelled

• Hemagglutinin H5 positive control template (for Standard curve RED)

• Neuramidase N1 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

• H5 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 Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

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 H5N1 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 Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

5

Principles of the test

The kit contains two primer and probe sets. The H5 primer and probe set are designed to
detect all influenza H5 sequences from the avian H5N1 lineage. The N1 primer and
probe set detect all N1 sequences regardless of the species of origin. Results from both
primer sets can be used to make a positive determination of the H5N1 genotype.
Samples that test negative for H5 and positive for N1 represent alternative circulating
subtypes that contain the N1 gene .

Real-time PCR

A H5N1 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 H5N1 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
H5N1 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 H5N1 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 false-positive 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 Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

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 H5N1 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 H5N1 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 Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

7

Component - resuspend in water

Volume

H5 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

H5 Positive Control Template (RED) *

N1 Positive Control Template (RED) *

500 µl

Quantification of Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

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

H5 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 Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

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

H5 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 Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

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 Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

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 H5N1 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 Influenza A virus subtype H5N1 (avian influenza) genomes

Advanced kit handbook HB10.05.09

Published Date: 20/09/2016

12