Influenza A/B virus detection and influenza A virus subtyping with emphasis on the novel H7N9 virus by using multiplex real-time RT-PCR

•Three sets of multiplex or singular real time RT-PCR platforms were established for differentially detecting the recent highly pathogenic H7N9, 2009 pandemic H1N1, seasonal H3, and avian H5 genomes.•The platforms effectively exclude the false positive cases from other respiratory infection agents.•The sensitivity for detecting the H7 and N9 genes was 5 copies per reaction, and analyzing clinical specimens by using this platform required only 3 h.

Infections of the novel avian influenza A H7N9 virus cause severe respiratory diseases and death. In this study, to develop highly sensitive methods for differentially detecting the H7N9 virus, multiplex and singular real-time reverse transcription polymerase chain reaction (RT-PCR) assays were established and examined by targeting the H7 and N9 genes of the H7N9 virus. Furthermore, an additional multiplex assay combining previous real time RT-PCR designs was established to subtype the pandemic H1N1, H3, and H5 influenza viruses. Applying the proposed assay system to analyze 100 clinical specimens collected from respiratory infection cases identified influenza A viruses (pandemic H1N1 and H3) in 23 samples. It has been demonstrated that other common respiratory viruses will not be detected by using this platform.