小反刍兽疫病毒液滴式数字 PCR 检测方法的建立及应用

苏佳，赵炜，刘伟洁，白洪旭，陈延飞，孙淼，吴华伟，薛青红，陈晓春

Abstract

为实现小反刍兽疫病毒（Peste des petits ruminants virus, PPRV）的快速、准确、定量检测，以国内 PPRV Clone 9 疫苗株N基因为靶位点设计特异性引物及探针，建立液滴式数字 PCR（Droplet digital PCR, ddPCR）方法，并评价其特异性、敏感性及重复性。对比该方法与实时荧光定量 PCR（Fluorescence quantitative PCR, qPCR）的灵敏度，同时用于 PPRV（Clone 9 株）核酸标准物质定量检测。结果显示，该 ddPCR 方法特异性良好，仅 PPRV 检测呈阳性，其余羊源病毒制品及毒株检测均为阴性；检测灵敏度优异，最低检出限为4.33 拷贝 /μL，相较 qPCR 方法57.3 拷贝 /μL的检出限，灵敏度高出 10 倍；重复性可靠，试验变异系数为1.8 %；定量结果精准，9 家具备资质单位的检测数据组间变异系数均小于5 %。本方法可高效完成 PPRV 核酸快速检测，可为该病毒临床诊断与核酸绝对定量提供可靠技术手段。

In order to achieve rapid, accurate and quantitative detection of Peste des petits ruminants virus (PPRV), a droplet digital PCR (ddPCR) method was established with specific primers and probe targeting the N gene of the domestic vaccine strain PPRV Clone 9, and the specificity, sensitivity and repeatability of the method were evaluated. At the same time, the sensitivity of the established ddPCR method was compared with that of the real-time fluorescence quantitative PCR (qPCR) method. Finally, the established ddPCR method was applied to the quantification of PPRV (Clone 9 strain) ribonucleic acid reference material. The results showed that the ddPCR method had good specificity, with only positive results for PPRV detection and negative results for other sheep-derived virus biological products and viruses. The sensitivity of the ddPCR method was high, with a detection limit of 4.33 copies/μL, which was 10 times higher than the sensitivity of the qPCR method (57.3 copies/μL). The coefficient of variation of the repeatability test was 1.8 % in the ddPCR method. Besides, the ddPCR method was accurate in quantification, as the coefficient of variation was less than 5 % between groups of measurement data in the nine qualified institutions. The ddPCR method established in this study can effectively and rapidly detect PPRV nucleic acid, providing an effective method for the diagnosis and absolute quantification of PPRV.