PEDV-TGEV-RV三重PCR检测方法的建立  

Establishment of multiplex PCR for simultaneous detection of porcine epidemic diarrhea virus, porcine transmissible gastroenteritis virus and porcine rotavirus

作　　者：邹云婧 梁中银[2] 林密[3] 袁万哲[1,4,5] 孙继国[1,4,5] ZOU Yun-jing LIANG Zhong-yin LIN Mi YUAN Wan-zhe SUN Ji-guo(College of Veterinary Medicine ,Hebei Agricultural University,Baoding 071001, China College ofA nimal Science and Technology ,Hebei Agricultural University ,Baoding 071001, China Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences ,Lanzhou 730046, China Hebei Engineering and Technology Research Center of Veterinary Biotechnology ,Baoding 071001, China North China Research Center of Animal Epidemic Pathogen Biology, the A gric ultural Minis try of China, Baoding 071001, China)

机构地区：[1]河北农业大学动物医学院,河北保定071001 [2]河北农业大学动物科技学院,河北保定071001 [3]中国农业科学院兰州兽医研究所,甘肃兰州730046 [4]河北省兽医生物技术工程技术研究中心,河北保定071001 [5]农业部动物疫病病原生物学华北区观测实验站,河北保定071001

出　　处：《中国兽医科学》2016年第11期1383-1387,共5页Chinese Veterinary Science

基　　金：河北省科技计划项目(16226604D)

摘　　要：根据G en Bank中已登陆的猪流行性腹泻病毒(PED V)、猪传染性胃肠炎病毒(TGEV)和猪轮状病毒(RV)的参考基因序列,设计3对引物分别用于扩增PEDV的N基因、TGEV的M基因、RV的VP7基因的目的片段;通过优化反应体系,建立了同时检测PEDV、TGEV、RV的三重PCR检测方法。敏感性和特异性试验的结果表明,该方法对这3种病毒的最低核酸检出量分别为39.5 pg(PEDV)、38.1 pg(TGEV)和51.6 pg(RV)。该方法可同时扩增477 bp(PEDV)、263 bp(TGEV)、355 bp(RV)的特异性基因片段,而对猪瘟病毒(CSF V)、猪繁殖与呼吸综合征病毒(PRRSV)、猪圆环病毒2型(PCV2)和伪狂犬病病毒(PRV)等病毒的检测结果均为阴性。三重PCR与单一PCR的符合率为100%。上述结果表明,该方法的建立对于这3种病毒的临床快速检测与流行病学调查具有十分重要的意义。According to the published relative sequences in GenBank of porcine epidemic diarrhea virus（PEDV）, porcine transmissible gastroenteritis virus（TGEV）,and porcine rotavirus（RV）,three pairs of specific primers were designed and synthesized to amplify the three specific fragments,including the N gene of PEDV,the M gene of TGEV and the VP7 gene of RV,respectively. A multiplex PCR was developed for simultaneous detection of PEDV, TGEV and RV after optimizing the annealing temperature and primer. The sensitivity and specificity tests showed that the multiplex PCR system was so sensitive that the minimum detectable amounts of RNA for PEDV,TGEV and RV could be 39.5 pg,38.1 pg,and 51.6 pg,respective- ly. Using this multiplex PCR system, specific PCR products of 477 bp for PEDV, 263 bp for TGEV and 355 bpfor RV could be simultaneously amplified ,and the testing results for CSFV, PCV2 and PRV were all nega- tive. By contrast,the two testing methods showed a.consistency of 100%. In conclusion, the multiplex PCR method could be used for the effective detection and diagnosis of those three virus.

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]