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Background   Influenza A virus is a leading causative pathogen of human acute respiratory infection. Recently, the co-circulation of pandemic (H1N1) 2009 and seasonal H3N2 viruses was reported, and sporadic cases with reassortant avian H7N9 virus are continually reported in China. We aimed to establish a multiplex one-step real-time reverse transcription-polymerase chain reaction (rRT-PCR) assay to simultaneously detect and discriminate FluA subtypes, including human seasonal H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus, in one reaction tube.    Methods  Clinical samples, including throat swabs and sputum, were collected from the patients with influenza-like illness (ILIs). Total viral RNA from each sample or viral culture was extracted, and the specific detection of FluA virus and its subtypes was performed using a multiplex rRT-PCR assay.    Results  The limitation of detection (LOD) of the multiplex assay was 5.4 × 10 −2  50% tissue culture infective dose (TCID 50 ) per reaction or 4.8 × 10 1  copies per reaction for each virus of the three viruses. For simultaneously detecting the three viruses, the LOD was 1.8 × 10 −2  TCID 50  per reaction or 1.6 × 10 copies per reaction for testing the total FluA virus RNA and 5.6 × 10 −2  TCID 50  per reaction or 5.1 × 10 copies per reaction for the H3, H1, and H7 genes in one reaction tube. The multiplex assay specifically detected these viruses, and no cross-reaction with other pathogens was found. Moreover, the assay had reliable clinical sensitivity (100%) and valuable clinical specificity (>95%). The detection of FluA with the matrix (M) gene contributed to the further determination of these subtypes, and the Rnase P gene (RP) was considered an internal control to favourably evaluate the quality of the clinical samples.    Conclusions  These findings indicate that the multiplex assay can simultaneously detect and discriminate FluA subtypes with reliable sensitivity and specificity, which is required for the early clinical diagnosis and viral surveillance of patients with FluA infection.

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Simultaneous detection of influenza A subtypes of H3N2 virus, pandemic (H1N1) 2009 virus and reassortant avian H7N9 virus in humans by multiplex one-step real-time RT-PCR assay