SYBR GreenⅠ RT-PCR for rapid diagnosis of H9 Subtype Avian Influenza Virus

A pair of specific primers targeting hemagglutinin gene of H9 avian influenza virus(AIV) was designed for the development of a SYBR Green I fluorescent based real-time RT-PCR(RRT-PCR) to quantify the H9 subtype AIV for rapid diagnosis.The detection limit of RRT-PCR was 8.33×102 plasmid copies.The melting curve analysis using SYBR Green I dye showed one specific peak at the melting temperature of(83.83±0.22)℃ with no primer-dimers peak.No amplification was detected by this method from the unrelated RNA samples,such as H5 subtype AIV,duck hepatitis virus,duck plague virus or avian paramyxovirus type-1.High reproducibility was obtained for detecting plasmid DNA with intra-assay of 0.52%-1.48% and inter-assay of 0.71%-2.21%.