Developing SYBR Green Ⅰ Real-Time PCR Assay For Rapid Detection Of Novel Influenza A H1N1and Seasonal Influenza Virus

Objective:To develop a SYBR Green I real-time PCR and a multiplex PCR assay to rapidly detect human novel H1N1virus, seasonal H1N1、H3N2and influenza B virus.Method:1. Primers were designed and selected from highly conserved regions of hemagglutinin (HA) gene of the four influenza virus, the four virus were detected by SYBR Green Ⅰ-based real-time fluorescence polymerase chain reaction with the analysis of dissociation curve (DC) and melting temperature(Tm), respectively. A primer specific for the GAPDH gene was added as a internal control to ascertain the source of sample and implement quality control.2. A multiplex PCR for typing and subtyping the four human influenza viruses were established. Specificity and sensitivity of the assay were assessed. Blind samples were detected with the multiplex-PCR.Result:1. The standard curves of SYBR Green I real-time PCR showed fine linear relationship between threshold cycle and template concentration, the amplification efficiency was95.588%and detective limit of the assay was101copies/reaction. It took only3.5hours from viral RNA extraction to PCR completion and of good reproducibility. The results showed that the assay had no cross-reaction with H5, H7, H9subtype influenza virus and parainfluenza virus type1,2,3.32blind samples were detected successfully.2. The subtype-specific primers were designed to amplify HA fragments of106bp for novel influenza A virus,135bp for seasonal H1N1influenza virus,118bp for seasonal H3N2influenza virus,170bp for influenza B virus. The sensitivity of the multiplex PCR was evaluated by testing the novel H1N1strain (A/colifornia/07/2009), and the testing limit was102copies/reaction. Every primer only detect its corresponding virus,32blind specimens were detected specifically.Conclusion:1. The SYBR Green Ⅰ-based real-time fluorescence polymerase chain reaction is sensitive, low-cost, high-throughput, and suitable for subtyping of human influenza virus without fluorescence probe.2. The multiplex PCR combines both sensitivity and specificity for not only detect the novel human H1N1virus, but also monitor the human seasonal H1N1、H3N3and influenza B virus simultaneously. It can be used in identification and subtyping of influenza virus.