Establishment of SYBR GreenI Real-time RT-PCR for Rapid Diagnosis of H5 Subtype Avian Influenza Virus

A pair of specific primers target to hemagglutinin gene of H5 avian influenza virus(AIV) was designed and a SYBR GreenⅠ fluorescent based real-time RT-PCR (RRT-PCR) was developed for the quantitation of H5 subtype AIV. The detection limit of RRT-PCR was 2.1×102 plasmid copies. The melting curve analysis using SYBR GreenⅠ dye showed one specific peaked with a melting temperature (Tm) of (79.5±0.3) ℃,and no primer-dimers peak represent. H5N1 and H5N2 subtype AIV could rapidly determined. No amplification was detected from unrelated RNA samples by this method. Fine reproducibility was obtained for detecting plasmid DNA with intra-assay of 2.99% and inter-assay of 5.12%.