H9亚型鸭源禽流感病毒SYBR GreenⅠ实时荧光定量RT-PCR快速检测方法的建立

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

针对H9亚型禽流感HA基因保守序列设计引物,建立基于SYBR GreenⅠ检测模式的荧光定量RT-PCR（real-ti me RT-PCR,RRT-PCR）,最低检测限为8.33×102拷贝质粒DNA,与常规PCR相比,灵敏度高出100倍。扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,Tm值为（83.83±0.22）℃,组内变异系数为0.52%~1.48%,组间变异系数为0.71%~2.21%,可重复性好;对H5亚型禽流感病毒、鸭肝炎病毒、鸭瘟病毒、鸭源禽1型副粘病毒无扩增;检测速度快,从样本处理到报告结果仅需4.5 h。

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%.