蓝舌病病毒小反刍兽疫病毒羊口疮病毒和山羊痘病毒多重荧光定量PCR检测方法的建立与应用

Establishment and preliminary application of multiplex real-time PCR for detecting bluetongue virus,peste des petits ruminants virus,orf virus and capripoxvirus

为建立一种能够同时检测蓝舌病病毒(BTV)、小反刍兽疫病毒(PPRV)、羊口疮病毒(ORFV)和山羊痘病毒(CaPV)的方法,基于TaqMan探针技术,分别以NS3、N、F1L和P32为靶基因设计特异性探针和引物。以特异性引物扩增目的基因并构建重组质粒标准品。通过优化反应体系建立多重荧光定量PCR检测方法,并用此方法检测临床样品。结果显示,该多重荧光定量PCR标准曲线呈线性关系,对BTV、PPRV、ORFV和CaPV的扩增效率分别为100%、91%、91%、95%;该方法具有较高的灵敏性和特异性,对BTV、PPRV、ORFV和CaPV阳性质粒的最低检出量分别为17、12、8和9 copies/μL,与口蹄疫病毒、腐败梭菌、布鲁菌及弓形虫无交叉反应;其重复性较好,组间变异系数和组内变异系数均小于5%。对232份样品进行检测,用该多重荧光定量PCR分别检出9份BTV、3份PPRV、3份CaPV和24份ORFV阳性样品,而用常规PCR分别检出7份BTV、3份PPRV、0份CaPV、12份ORFV阳性样品。综上,该方法能够同时检测BTV、PPRV、ORFV和CaPV,可用于羊病的鉴别诊断、流行病学调查和疫病防控。

In order to establish a detection method that can simultaneously detect bluetongue virus(BTV),peste des petits ruminants virus(PPRV),orf virus(ORFV) and capripoxvirus(Ca PV),based on Taq Man probe technology,specific probes and primers were designed with NS3,N,F1L and P32 as target gene,respectively.The target genes were amplified with specific primers to construct recombinant plasmids.A multiplex real-time PCR assay was established by optimizing the reaction system,and this method was used for the detection of clinical samples.The results showed that the multiplex real-time PCR standard curves were linear,and the amplification efficiencies of BTV,PPRV,ORFV and Ca PV were100%,91%,91%,and 95%,respectively.This method performed well with high sensitivity and specificity.The minimum detection amounts of positive plasmids for BTV,PPRV,ORFV and Ca PV were 17 copies/μL,12 copies/μL,8 copies/μL,9 copies/μL,respectively.There was no cross-reaction with foot-and-mouth disease virus,Clostridium septicum,Brucella and Toxoplasma gondii and the coefficient of variation between inter-groups and intra-groups were both less than 5%.Total 232 samples were tested by the multiplex real-time PCR and conventional PCR.The results showed that 9 BTV positive samples,3 PPRV positive samples,3 Ca PV positive samples,24 ORFV positive samples were respectively detected by the multiplex real-time PCR. However,conventional PCR only detected 7 BTV positive samples,3 PPRV positive samples,0 Ca PV positive samples,12 ORFV positive samples,respectively.Therefore,this multiplex realtime PCR method is able to detect four main goat and sheep viruses in one reaction with good specificity and sensitivity and could be used for differential diagnosis,epidemiological investigation and epidemic prevention and control of goat and sheep diseases.