猪四种病毒性腹泻病原多重PCR检测方法的建立及应用  

Establishment and application of the multiplex PCR for detection of four porcine viral diarrhea viruses

作　　者：熊年年 陶洁[1,3] 张清真[1,2] 张春玲[1,3] 李本强[1,3] 常宏赏 刘惠莉[1,3] 

机构地区：[1]上海市农业科学院畜牧兽医研究所,上海201106 [2]上海海洋大学水产与生命学院,上海201306 [3]上海市农业遗传育种重点实验室,上海201106

出　　处：《中国兽医科学》2016年第8期972-978,共7页Chinese Veterinary Science

基　　金：上海市农委重点攻关项目[沪农科攻字(2013)第5-5号;沪农科攻字(2013)第3-6号];上海市扬帆计划项目(15YF1410300)

摘　　要：分别以猪流行性腹泻病毒(PEDV)M基因、猪轮状病毒(Po RV)VP6基因、猪传染性胃肠炎病毒(TGEV)N基因、猪源牛病毒性腹泻病毒(BVDV)Npro基因为靶基因,参照Gen Bank上登录的基因序列,利用DNAStar和Oligo7软件设计了4对引物。采用PCR分别扩增各病毒基因,并克隆至p MD18-T载体,获得4个阳性重组质粒。以重组质粒为模板,进行多重PCR方法的优化。特异性试验检测可分别扩增出相应的病毒基因片段;敏感性试验结果显示,检测PEDV、TGEV、Po RV及BVDV质粒的最低拷贝数分别为730copies/L、547 copies/L、6.47×103copies/L、705 copies/L。应用该方法对猪场44份猪腹泻样品进行检测,结果检出15份PEDV样品,3份TGEV样品,2份BVDV样品,其中PEDV与TGEV混合感染样品2份,TGEV与BVDV混合感染样品1份。随机抽取其中2份PEDV阳性样品进行病毒分离,病毒经细胞传3代以上PCR检测均为阳性,对PCR产物测序证实为PEDV。结果表明,本研究建立的多重PCR方法具有快速、简便、特异性强、敏感度高的特点,能够对PEDV、Po RV、TGEV和BVDV单个或混合感染的临床样品进行快速鉴别诊断。Four pairs of primers against PEDV M gene, TGEV N gene, PoRVVP6 gene and BVDV Npro genes were designed using DNASTAR and 01igo 7 software. After PCR amplification, the four genes were cloned into pMD18 T vector. Then the multiplex PCR method was optimized using the recombinant plasmids as templates. Specificity experiments showed that each viral gene fragment could be amplified successfully. Sensitivity test indicated that the minimum copy numbers of the plasmids contained PEBV,TGEV,PoRVand BVDV geneswere730copies/μL, 547eopies/μL, 6.47×10^3copies/μL, 705 copies/μL, respectively. 44 swine diarrhea samples were collected and detected using this method. Results showed that there were fifteen PEDV positive samples, three TGEV positive samples and two BVDV positive samples. Besides, there were two samples co-infected with PEDV and TGEV, and one sample co-infected with TGEV and BVDV. Further more the two PEDV positive samples were inoculated on cells for virus isolation and the results confirmed that the three generations of cell cultures were PEDV positive by PCR combined with sequencing. In con- clusion, the multiplex PCR method established in this study is convenient, high specificity and sensitivity which will be helpful for the rapid differential diagnosis of PEDV, PoRV, TGEV and BVDV.

关 键 词：猪病毒性腹泻病原 多重PCR检测技术 PEDV TGEV Po RV BVDV 

分 类 号：S852.651[农业科学—基础兽医学]