西尼罗病毒荧光定量PCR检测体系的建立及评价  

Establishment and Evaluation of Real-time PCR forWest nile virus Detection

作　　者：史利军[1,2] 吕茂民[3] 孙卫华[3] 尹惠琼[3] 黎诚耀[4] 章金刚[1,2] 

机构地区：[1]华中农业大学动物医学院 [2]军事医学科学院野战输血研究所,北京100850 [3]军事医学科学院野战输血研究所 [4]南方医科大学生物技术学院

出　　处：《农业生物技术学报》2008年第5期748-751,共4页Journal of Agricultural Biotechnology

基　　金：全军医药卫生科研基金项目(No.06H056)资助

摘　　要：建立了一种实时荧光定量PCR快速检测西尼罗病毒(West nile virus,WNV)的方法。通过序列比对和blast分析,确定WNV Caspid蛋白保守区基因为检测的目的基因,引物采用Primer Premier5.0软件设计。利用成本相对较低的SYBR GreenⅠ染料法建立WNV核酸检测方法。通过对标准品的扩增、测序及对标准扩增曲线的绘制,建立WNV核酸检测体系。同时对建立的检测体系进行了检测特异性、灵敏度和重复性分析。将阳性对照标准品进行10倍梯度稀释后可检测到102copies/μL样品,表明该检测体系具有较高的检测灵敏度。通过分析表明,本检测体系在用空白对照及类似的乙脑病毒作为扩增对照时,没有发现非特异性产物的生成,表明该体系对于WNV的检测是特异的。通过4次批间重复检测,体系的变异系数小于2%,表明该检测体系具有良好的可重复性。A rapid Real-time polymerase chain reaction （RT-PCR） for West nile virus（WNV） detection was established. Primers were designed according to Capsid protein gene by Primer Premier5.0. In response, a cheap assay with the intercalating dye SYBR green Ⅰ was developed and validated. Amplifying curve showed that this method could successfully amplify 10^2 copies/μL WNV gene, meanwhile reference Japanese encephalitis virus （JEV） and blank control were all negative. Ten-fold successive dilutions of positive WNV DNA were used to measure the sensitivity of RT-PCR. The assay showed high reproducibility with CV〈2%, indicating that the developed RT-PCR assay, a rapid, sensitive and specific test, has been built for detecting WNV.

关 键 词：西尼罗病毒 Caspid蛋白基因 实时荧光定量PCR 

分 类 号：S188[农业科学—农业基础科学]