小反刍兽疫病毒液滴式数字PCR检测方法的建立及应用

Establishment and Application of a Droplet Digital PCR Assay for Detection of Peste des Petits Ruminants Virus

关键词:  小反刍兽疫病毒  液滴式数字PCR  荧光定量PCR  检测

Key words:  Peste des petits ruminants virus  droplet digital PCR  fluorescence quantitative PCR  detection

摘要:

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，建立了液滴式数字PCR(Droplet digital PCR，ddPCR)方法，并对其特异性、敏感性及重复性进行评价。将所建立的ddPCR方法与实时荧光定量PCR(Fluorescence quantitative PCR，qPCR)方法的灵敏度进行比较分析并应用于PPRV(Clone 9株)核糖核酸标准物质的定量。结果显示，所建立的ddPCR方法特异性好，仅PPRV检测结果为阳性，其他羊源病毒类生物制品及毒种检测结果均为阴性；敏感性高，基因拷贝数检出限度为4.33拷贝/μL，比qPCR方法的灵敏度(57.3拷贝/μL)高10倍；重复性好，重复性试验的变异系数为1.8%；定量准确，具有一定资质的9家单位各组测量数据组间变异系数小于5%。试验建立的ddPCR方法能够有效地对PPRV核酸进行快速检测，为PPRV的诊断及绝对定量提供了有效方法。

Abstract:

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.