实时荧光PCR技术快速定量检测H5亚型禽流感病毒

Real-time fluorescent PCR in the rapid detection of H5 subtype avian influenza virus

选取H5亚型禽流感病毒血凝素(Hemagglutinin,HA)基因保守序列,使用Primer Express 2.0软件设计出特异性引物和TaqMan MGB探针,利用实时荧光PCR技术来定量检测禽流感病毒。使用含有选定检测序列的RNA标准品做标准曲线,结果表明该方法的灵敏度为10拷贝/反应,标准曲线的相关系数为0.998003,对H9亚型禽流感病毒和其他禽病病毒无交叉反应,特异性好、重复性佳。为禽流感病毒检测提供了一种特异、敏感、快速的定量检测方法。

Real-time fluorescent PCR for detection of H5 subtype AIV was developed using specific primers. TaqMan MGB probes were designed with Primer Express 2.0 software according to the conserved region of the H5 subtype AIV hemagglutinin gene. A serial dilutions of H5 KNA standard was prepared by ha vitro transcription and used for generating standard curve. The results showed that the real-time PCR was capable of detecting 10 copies of HA genes/reaction, with a correlation coefficient of 0.998003. No cross-reaction was detected against other avian disease viruses. This method was highly specific and sensitive, and could be used for rapid quanfitatve detection of H5 subtype avian influenza virus.