Simultaneous detection of influenza A viruses and 2009 pandemic influenza A(H1N1) virus by multiplex real-time RT-PCR

Objective To establish a TaqMan-based multiplex real-time PCR assay for the rapid and simultaneous detection of influenza A viruses and 2009 pandemic influenza A(H1N1) virus.Methods The specific primers and probes were designed in the conserved region of the M gene for influenza A viruses and the NA gene for 2009 pandemic influenza A(H1N1) virus,respectively.The reaction conditions were optimized and the sensitivity,specificity and stability of the assay were evaluated.The clinical specimens collected from the patients were detected by this assay.Results The results showed that the assay possessed high specificity for influenza A viruses and 2009 pandemic influenza A(H1N1) virus detection and the detection limit was up to 0.01 TCID50 and 0.1 TCID50 respectively.The viral RNA could be directly detected from the clinical specimens by this assay.Conclusion The multiplex real-time RT-PCR assay can provide rapid,sensitive and reliable detection of influenza A viruses and 2009 pandemic influenza A(H1N1) virus and is useful in the detection of influenza virus.