同时检测禽流感和新城疫病毒的一步法荧光定量RT-PCR方法的建立

The Establishment of one step fluorescent RT-PCR method for simultaneous detection of the AIV and NDV

分别根据新城疫病毒与禽流感病毒的M基因、禽流感病毒HA基因上的保守序列,设计了通用型禽流感病毒、H5\H7\H9亚型及通用型新城疫病毒5对特异性引物和5条Taqman荧光标记核酸探针。以阳性重组质粒进行体外转录后的RNA为标准品绘制标准曲线,建立了一步实时荧光RT-PCR方法。结果表明,本试验建立的标准曲线Ct值与模板浓度具有良好的线性关系,相关系数大于0.99。通过利用所建立的方法与鸡胚分离法同时对新鲜采集的临床样品进行检测,2种方法的检测结果符合率为100%,说明在临床样品检测、流行病学监测方面具有良好的应用前景。

In order to integrated rapid detect avian influenza virus and Newcastle disease virus that seriously harm to the development of the poultry industry, we respectively designed five pairs of special primers and five Taqman probes of universal influenza virus,H5／H7／H9 subtype and uni- versal NDV that based on conserved sequences on HA gene of AIV and the M gene of NDV and AIVo We established one step real-time fluorescent RT-PCR that with positive recombinant plas- mid in vitro transcription of RNA as a standard for standard curve. The results showed that the standard curve was established in this experiment, The Ct value has a good linear relation with the template concentration,the CV is larger than 0.99. By using the established method with virus i- solation method to detect the clinical samples collected freshly, compliance of the two method was 100%,indicating that established method nasa good application prospect in clinical sample detec- tion and epidemiological monitoring.