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