PEDV、TGEV和PoRV三重PCR检测方法的建立及初步应用  

Establishment and Preliminary Application of Triplex PCR for Simultaneous Detection of PEDV,TGEV and PRV

作　　者：蒲翠敏 冯旭芳 张双翔[3] 胡兴义 张海[1] 丁尊俄 王开功[1] 周碧君[1] 文明[1] 程振涛 

机构地区：[1]贵州大学动物科学学院,贵州贵阳550025 [2]贵州省德江县动物疫病预防控制中心,贵州德江565200 [3]贵州省动物疫病预防控制中心,贵州贵阳550001

出　　处：《动物医学进展》2018年第2期19-25,共7页Progress In Veterinary Medicine

基　　金：贵州省科学技术厅农业攻关项目(黔科合NY[2015]3009-1号);贵州省生猪质量安全工程技术研究中心建设项目(黔科合农C字[2011]4022号);贵州省动物疫病防控与兽医公共卫生保障科技创新人才团队项目(黔科合人才团队[2015]4016号);贵州省农业委员会科技计划项目[2016-01号]

摘　　要：为建立一种快速、敏感的猪流行性腹泻病毒(PEDV)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(PoRV)三重PCR检测方法,根据GenBank登录的PEDV S基因、TGEV M基因和PoRV VP7基因参考序列,设计3对特异性引物,以克隆的质粒为模板,进行反应条件的优化以及特异性、敏感性试验。结果采用所建立的方法能够扩增出PEDV、TGEV和PoRV特异性片段,且不能检出PCV2、CSFV、PRRSV;对PEDV、TGEV和PoRV质粒模板的最低检测量分别为1×10~2拷贝/μL、1×100拷贝μL、1×10~3拷贝/μL;对10~2份临床病料进行检测,结果 PEDV、TGEV和PoRV的检出率分别为37.25%、1.96%和6.86%,与单项PCR检测结果进行比较,符合率均为100%。结果表明,所建立的三重PCR检测方法可为猪病毒性腹泻病的快速诊断及流行病学调查提供技术支持。To establish a rapid and sensitive triplex PCR method for detection of porcine epidemic diarrheavirus （PEDV）,porcine transmissible gastroenteritis virus （TGEV） and porcine rotavirus （PoRV） simulta-neously,according to the reference sequences of PEDV S gene,TGEV M gene and PoRV VP7 gene in Gen-Bank,three specific primers were designed, the cloned plasmids were used as templates to optimize the reac-tion conditions, and specificity and sensitivity tests were conducted. The specific fragments of PEDV,TGEV and PoRV could be amplified by this method, and PCV2, CSFV, PRRSV couldn＇t be detected. Thedetection limits of PEDV,TGEV and PRoW were 1.41 X 10^2 , 1.41 X 102 and 1.41 X 103 copies/μL, respec-tively. Besides, 102 clinical samples were detected and the detection rates of PEDV,TGEV and PoRV were37.25% ,1.96% and 6.86% ,which were consistent with the result using the single PCR,and the coinci-dence rate was 100 %. The results showed that of established triplex PCR could provide effective technicalsupport for the rapid diagnosis and epidemiological investigation of swine viral diarrhea pathogens.

关 键 词：猪流行性腹泻病毒 猪传染性胃肠炎病毒 猪轮状病毒 三重PCR 

分 类 号：S852.651[农业科学—基础兽医学]