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Hemagglutinin (HA) gene &
Neuraminidase (NA) gene
H10N8
PCRmax Ltd qPCR test
TM
150 tests
For general laboratory and research use only
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Quantification of H10N8 genomes
Advanced kit handbook HB10.01.09
Published Date: 13/10/2016
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Avian Influenza (‘avian flu’ or ‘bird flu’) strains all belong to the Influenza A virus which are
generally adapted to birds but not exclusive to them.
Of particular concern to humans are ones which are infectious to both humans and birds.
Specific strains such as H1N1 have been the subject of much media concern and
speculation over pandemics and its widespread transmission globally.
Only some strains of avian influenza are pathogenic in humans typically H5N1, H7N3,
H7N7 and H7N9 but now H10N8 has proven fatal in China as of Dec 2013.
Influenza type A viruses are 80–120 nanometers in diameter and usually roughly spherical,
made up of a viral envelope containing two main types of proteins, wrapped around a
central core.
The two large proteins found on the outside of viral particles arehemagglutinin
(HA)andneuraminidase (NA). HA is a protein that mediates binding of the virion to target
cells and entry of the viral genome into the target cell, while NA is involved in the release of
progeny virions from infected cells
Influenza type A viruses are categorized into subtypes based on the type of these two
proteins on the surface of the viral envelope.
The central core of a virion contains the viral genome and other viral proteins that package
and protect the genetic material
The entire Influenza A virus genome is ~13,588 bases long and is contained on8RNA
segments that code for11proteins.
Introduction to H10N8
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Quantification of H10N8 genomes
Advanced kit handbook HB10.01.09
Published Date: 13/10/2016
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Specificity
The PCRmax™ qPCR Kit for H10N8 (H10N8) genomes is designed for the in vitro
quantification of H10N8 genomes. The kit is designed to have the broadest detection
profile possible whilst remaining specific to the H10N8 genome.
The primers and probe sequences in this kit have 100% homology with a broad range of
H10N8 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.
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Quantification of H10N8 genomes
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Kit Contents
• H10N8 specific primer/probe mix (150 reactions BROWN)
FAM labelled
• H10N8 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
• H10N8/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 template 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.
Lyophilised OneStep 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
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Quantification of H10N8 genomes
Advanced kit handbook HB10.01.09
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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 H10N8 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.
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Quantification of H10N8 genomes
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Principles of the test
Real-time PCR
A H10N8 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 H10N8 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 H10N8 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 H10N8 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.
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Quantification of H10N8 genomes
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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 H10N8 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 H10N8 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-N-glycosylase (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.
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Quantification of H10N8 genomes
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Component - resuspend in water
Volume
H10N8 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
H10N8 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.
3. Reconstitute the positive control template in the template preparation buffer
supplied, according to the table below:
To ensure complete resuspension, vortex this tube thoroughly.
500 µl
Post-PCR heat-sealed foil
Component - resuspend in template preparation buffer
Volume
H10N8 Positive Control Template (RED) *
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Quantification of H10N8 genomes
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One Step RT-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
H10N8 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.
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Quantification of H10N8 genomes
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One Step Amplification Protocol
Amplification conditions using Lyophilised OneStep 2x RT-qPCR Mastermix.
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.
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 x50
* Fluorogenic data should be collected during this step through the FAM and VIC channels
Component
Volume
Lyophilised OneStep 2x RT-qPCR Mastermix
1 µl
H10N8 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:
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Quantification of H10N8 genomes
Advanced kit handbook HB10.01.09
Published Date: 13/10/2016
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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.
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Quantification of H10N8 genomes
Advanced kit handbook HB10.01.09
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Internal PCR control
The Cq 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. Cq values of 28±3 are within the normal range. When
amplifying a H10N8 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.
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Quantification of H10N8 genomes
Advanced kit handbook HB10.01.09
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