野生型非洲猪瘟病毒多重数字PCR方法的建立

Establishment of a Multiplex Digital PCR Method for Wild African Swine Fever Virus

为建立一种鉴别诊断野生株和基因缺失株非洲猪瘟病毒(African swine fever virus,ASFV)的方法,针对ASFV的B646L、EP402R、DP96R基因保守序列设计3套引物和探针,优化三重荧光定量PCR体系作为基础建立微滴式数字PCR(droplet digital PCR,dd PCR)检测方法,并对方法的特异性、灵敏性及重复性进行评估。结果显示,优化后的多重荧光定量PCR检测方法的标准曲线线性关系良好,对PRRSV、PEDV、PRV、VSV、PCV、RNase-free水、重组质粒标准品进行特异性检测,特异性良好;重复性试验结果显示,变异系数均小于2%;对3种重组质粒的最低检测下限均为102 copies/μL。参照上述多重荧光定量PCR方法优化后的条件,进行多重ddPCR检测方法的建立,并且对该方法进行评价。结果显示,ddPCR检测方法标准曲线的线性关系良好;特异性及重复性良好;最低检测下限B646L为11.6 copies/μL、EP402R为13.3 copies/μL、DP96R为16.9 copies/μL;ddPCR的检测灵敏度比荧光定量PCR有优势。使用建立的多重ddPCR方法对14份P3实验室中的样品进行实验室样品检测,样品检测符合率达到100%。成功建立了能够同时检测用于鉴别野毒株感染与疫苗株的荧光定量PCR和微滴式ddPCR方法。

To establish a method for the differential diagnosis of African swine fever virus(ASFV)wild strain and gene deletion strain,three sets of primers and probes were designed for the conserved sequences of ASFV B646L,EP402R and DP96R genes.A droplet digital PCR(ddPCR)detection method based on the optimization of triplex real-time PCR system was established,and the specificity,sensitivity and repeatability of the method were evaluated.The results showed that the standard curve of the optimized multiplex fluorescent quantitative PCR detection method had a good linear relationship,and the specificity of PRRSV,PEDV,PRV,VSV,PCV,RNase-free water and recombinant plasmid standards was good.The results of repeatability test showed that the coefficient of variation was less than 2%.The lowest detection limit of the three recombinant plasmids was 102 copies/μL.According to the optimized conditions of the multiplex real-time PCR method mentioned above,the multiplex ddPCR detection method was established and evaluated.The results showed that the standard curve of ddPCR detection method had good linear relationship,specificity and repeatability.The lowest detection limit was 11.6 copies/μL for B646L,13.3 copies/μL for EP402R and 16.9 copies/μL for DP96R.The detection sensitivity of ddPCR is better than that of fluorescent quantitative PCR.Laboratory samples from 14 P3 laboratories were tested using the established multiplex ddPCR method.The coincidence rate of sample detection was 100%.The real-time PCR and droplet ddPCR methods for simultaneous detection of wild-type and vaccine strains of ASFV were successfully established,with high sensitivity,repeatability and specificity.