非洲猪瘟病毒三重荧光定量PCR鉴别检测方法的建立及应用

Development and application of a triplex real-time PCR method for detection and differentiation of African swine fever virus

为建立一种可以同时鉴别ASFV基因Ⅱ型强毒株、基因Ⅰ型弱毒株、I177L基因缺失疫苗毒株的TaqMan三重实时荧光定量PCR检测方法,设计了针对B646L、F778R和I177L基因的特异性引物和探针,并对该方法进行特异性、灵敏性和重复性试验检测,绘制标准曲线,最后用该方法和WOAH推荐的方法对临床样品进行检测和分型。结果显示,本方法特异性和灵敏性好,对B646L、F778R和I177L阳性质粒的最低检测下限分别为1、10和1 copies/μL,说明本方法敏感性高;且该方法在1×10^(0)copies/μL~1×10^(7)copies/μL模板范围内具有良好的线性关系;组内和组间变异系数均小于2%,重复性和稳定性良好。利用本方法和WOAH推荐的方法分别对临床样品进行检测,两种方法的ASFV阳性率和毒株分型结果一致。综上所述,本方法可以检测基因Ⅱ型强毒株、基因Ⅰ型弱毒株、I177L基因缺失疫苗毒株,为临床ASFV的检测和分型诊断提供了良好的技术支撑。

This study was aimed to develop a TaqMan triple Real-time PCR method for differential detecting genotype Ⅰ,genotype Ⅱ and I177L gene-deleted strain of African swine fever virus.Specific primers probes were designed based on the B646L,F778R and I177L genes.The specificity,sensitivity and repeatability of the triple Real-time PCR method were tested,and the standard curve was drawn.Finally,the triple Real-time PCR method and the method recommended by WOAH were used to detect and type the clinical samples.(Result)The method has strong specificity and sensitivity,the minimum detection limits of B646L,F778R and I177L positive plasmid were 1,10 and 1 copies/μL,respectively,with high sensitivity.The method had good linearity in the range from 1×10^(0)copies/μL~1×10^(7)copies/μL template.The reproducibility of intra-and inter-assay displayed that the coefficient of variation less than 2%,indicating that the method was repeatable and stable.Clinical samples were tested and typed in parallel by the method established in this study and the method recommended by wOAH.In this study,a triplex real-time PCR method was successfully established for the differential detection of ASFV genotype Ⅰ,genotype Ⅱ and I177L gene-deleted strain,which provides a good technical support for the clinical diagnosis and control of ASFv.