Influenza A Matrix Protein Gene M2

Real-time PCR kit using hybridization probes for the detection of Influenza A.

The common target for the detection of Influenza A is the matrix protein gene (M2). There are several publications describing the Real-Time-PCR detection adressing the viral M2 gene 1-4. This kit detects the M2 gene as a general test for Influenza A. The included internal control is equipped with the LC690 dye (since lot 596), allowing to run this kit on capillary LightCycler® instruments and on the LightCycler® 480 II system.

This latest version of the kit (lot 665) has been included in a clinical evalution. The detection limit was 8-15 genomic copies per reaction (3/3 triplicates positive). From a given panel of 16 human pathogen Influenza virus all has been detected positive. Clinical samples including H1N1/2009 samples were detected positive. Other viruses and bacteria were tested negative, confirming the specifictiy.

The Hemagglutinin (HA) and Neuraminidase (NA) types can be determined by specific PCR tests. The typical work flow will start with M2 gene testing and further analysis or typing of all Influenza A positive samples. In pandemic situations one might change the procedure and test M2 and the type specific assay simultanously (parallel) preferentially using a test for the HA type.

Influenza (flu) is a viral infection affecting the upper respiratory tract, causing a wide spectrum of symptoms, ranging from mild illness to more severe cases with caughing, high fever, general weakness and pneumonia, which can be fatal. The disease is commonly transmitted as aerosol through the air by coughs or sneezes and spreads around the world in seasonal epidemics typically during the winter season.

Influenza A is a negative strand ssRNA virus from the Orthomyxovirus family which infects birds and mammals. It is characterized by the Hemagglutinin (HA) and Neuraminidase (NA) genes. The Spanish flu 1918/19 was type H1N1 and has been made responsible for between 10 and 50 millions casualties. Some years ago a new Asian H5N1 bird flu caused several deaths and very recently a swine H1N1 virus from Mexico was suspected to have a pandemic potential.

All type A influenza viruses, including those that regularly cause seasonal epidemics of influenza in humans, are genetically labile and well adapted to elude host defenses. Influenza viruses lack mechanisms for the “proofreading” and repair of errors that occur during replication. As a result of these uncorrected errors, the genetic composition of the viruses changes as they replicate in humans and animals. This also results in the possibility that viruses of low pathogenicity can, after circulation for sometimes short periods in a host population, mutate into highly pathogenic viruses.

The matrix protein gene 2 (M2 or MP) is genetically stable and better conserved between different virus strains and the preferred target for RT-PCR based tests for Influenza A.

In addition we offer LightMix® Kits for the specific detection of the N1 gene, the H5 (Asia H5N1) gene, and for the H1 (swine H1N1 Mexico) gene, which can be used for the identification of specific strains.

We recommend to run a 2-step RT-PCR procedure, starting with generation of cDNA using the Roche Diagnostics Transcriptor kit and run then the PCR using the FastStart DNA master hybridization probes or the FastStart DNA master PLUS kit. A novel 1-step RNA master will be available May 2009.

1 Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement. Ward CL, Dempsey MH, Ring CJ, Kempson RE, Zhang L, Gor D, Snowden BW, Tisdale M. J Clin Virol. 2004 Mar;29(3):179-88

2 Application of a fluorogenic PCR assay for typing and subtyping of influenza viruses in respiratory samples. Schweiger B, Zadow I, Heckler R, Timm H, Pauli G. JCM 38 (2000) 1552-1558

3 Development of a real-time reverse transcriptase PCR assay for type A influenza virus and the avian H5 and H7 hemagglutinin subtypes. Spackman et al. JCM 40 (2002) 3256-3260

4 Simultaneous detection of influenza viruses A and B using real-time quantitative PCR. van Elden LJ, Nijhuis M, Schipper P, Schuurman R, van Loon AM. JCM 39 (2001) 196-200

Not for sale in USA.

History of versions

234 Original version optimized for teh detection of seasonal Influenza A including H5N1
596 New primer for H1N1 detection included (since April 27th, 2009)
662 Reverse primer changed to increase the sensitivity (since July 21th, 2009)
665 Adaption of the mixture of detection probes to achieve more balanced fluorescence levels for InfA H1N1 seasonal, H1N1/2009, H3N2, H5N1 (Aug 4th, 2009)

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