实时荧光定量RT-PCR方法检测禽流感病毒

根据禽流感病毒(Avian influenza virus,AIV)M基因上的保守序列,合成引物和荧光标记探针,以阳性AIVM基因质粒为标准品做标准曲线,建立了荧光定量逆转录聚合酶链反应(RRT-PCR)检测方法。结果表明,本试验建立的标准曲线循环阈值(Ct值)与模板浓度具有良好的线性关系,相关系数为0.999,灵敏度约为5拷贝/μL,相当于5个AIV颗粒,对新城疫病毒和其他禽病病毒无交叉反应,特异性好、重复性佳,为AIV检测提供了一种特异、敏感、快速的定量检测方法。对500份临床泄殖腔棉拭样品的检测,其结果阳性?阴性数与经典病毒分离方法符合率分别为91.2%?99.4%。在AIV临床样品筛检、流行病学监测等方面显示良好的应用前景。

Detection of avian influenza virus by real-time RT-PCR

According to the conservative region of AIV M gene, the primers and fluorescent probe were synthesized. A serial 10 fold dilutions positive plasmid was prepared and used for standard. The standard curve revealed the linear relationship between CT (cycle threshold) and template concentration. The RRT-PCR method for the detection of AIV was highly specific and sensitive and could be used for a rapid quantitative detection of AIV. No cross-reaction was detected against other avian disease viruses. Sensitivity was 5 copies of AIV genome. The meet rate of positive sum and negative sum with traditional virus isolation method was 91.2% as well as 99.4% in detecting 500 clinical cloacal swab samples,respectively.