Abstract:The purpose of this study was to develop a SYBR GreenⅠreal-time fluorescence quantitative PCR assay for rapid detection of novel influenza A H1N1 virus, seasonal H1N1, H3N2 and influenza B viruses. Specific primers were designed according to the conserved sequence of hemagglutinin (HA) gene of the four influenza viruses. The viruses were detected by SYBR GreenⅠreal-time fluorescence quantitative PCR with the analysis of dissociation curve (DC) and melting temperature(Tm), respectively. Sensitivity assay and reproducibility assay was determined. The results showed that the assay had no cross-reaction with H5, H7, H9 subtype influenza virus and parainfluenza virus type 1, 2, 3. The standard curves established by standard plasmid showed fine linear relationship between threshold cycle (Ct) and template concentration. The amplification efficiency was 95.588% and detection sensitivity was 101copies/reaction system. The results had fine repetition,and 32 blind samples were detected successfully. This SYBR Green Ⅰreal-time fluorescence quantitative PCR is sensitive, low-cost, high-throughput and suitable for subtyping of human influenza virus without fluorescence probe.