猪传染性胃肠炎病毒RT-PCR检测方法的建立及临床应用  

Establishment of a RT-PCR method for detection of transmissible gastroenteritis of swine virus and its clinical application

作　　者：王黎[1] 李碧[1] 周远成[1] 朱玲[1,2] 徐志文[1,2] 郭万柱[1,2] 

机构地区：[1]四川农业大学动物生物技术中心,四川雅安620514 [2]四川农业大学动物疫病与人类健康四川省重点实验室,四川雅安620514

出　　处：《中国兽医学报》2015年第2期190-194,共5页Chinese Journal of Veterinary Science

基　　金：四川省科技支撑计划资助项目(2012NZ0001)

摘　　要：根据Gen Bank登录的猪传染性胃肠炎病毒(TGEV)的N基因,针对其保守序列,设计并合成1对引物,可特异性扩增长度为590 bp目的条带,成功建立了检测TGEV的RT-PCR方法。以等量同一浓度的同一TGEV阳性病毒液进行重复性试验,其3次扩增特异性条带大小均为590 bp。以猪伪狂犬病病毒(PRV)、猪细小病毒(PPV),猪瘟病毒(CSFV)、猪蓝耳病病毒(PRRSV)、猪流行性腹泻病毒(PEDV)、猪轮状病毒(RV)进行特异性试验,其结果显示仅TGEV扩增得到预期特异性目的条带。以TGEV阳性病毒液提取的c DNA用紫外分析仪确定核酸质量浓度为1 g/L后,用dd H2O按10倍稀释,稀释度为10-1~10-5,其均能扩增出590 bp特异性条带。在猪传染性胃肠炎高发季节的2013年9月至2014年1月对四川成都、德阳、绵阳、遂宁、南充各地猪场送检的疑似猪传染性胃肠炎感染病猪的临床样品15份,进行检测,其猪传染性胃肠炎阳性率为100%。并且对四川成都、德阳、绵阳、遂宁、南充各地猪场保育舍随机采集的1~10周龄仔猪临床样品500份,进行检测,其1~2周龄仔猪传染性胃肠炎阳性率为17%,病死率为100%,3~4周龄仔猪传染性胃肠炎阳性率为14%,病死率为78.6%,5~6周龄仔猪传染性胃肠炎阳性率为9%,病死率为44.4%,7~8周龄仔猪传染性胃肠炎阳性率为4%,病死率为25%,9~10周龄仔猪传染性胃肠炎阳性率为1%,病死率为0%。从地区来看,在这500份样品中,成都、德阳、绵阳、遂宁、南充地区此病的阳性率分别为15%,6%,10%,6%,8%。以上结果显示所建立的TGEV RT-PCR检测方法,重复性好,特异性强,敏感性高,可用于该病的临床诊断。Based on TGEV N protein gene sequence recorded in Gen Bank,a pair of primers was uniquely designed to amplify a nucleotide length of 590 bp. And then,specificity test,sensitivity test and repeatability test were done to create a new method for diagnose of TGEV in clinic. The specificity test was operated by amplifying TGEV,PRV,PPV,CSFV,PRRSV,PEDV,RV respectively,and the results showed that only the TGEV sample could amplify a nucleotide of 590 bp. The sensitivity test used double distilled water to dilute 1 g / L c DNA of TGEV from 10- 1to 10- 5,and the lowest dilution 10- 5sample could be successfully detected by PCR. The repeatability test was done by amplifying the c DNA of TGEV three times,with an obvious consequence of 590 bp nucleotides. After the method was successfully established,we used this method to detect suspected TGEV infected piglet from several districts in Sichuan province. On the high incidence of transmissible gastroenteritis disease season,from September 2013 to January 2014,15 samples of piglet from Chengdu,Deyang,Mianyang,Suining,Nanchong were diagnosed as positive for TGEV. And we randomly collected 500 piglet samples from 1 week to 10 weeks to do a statistical analysis in those districts. By using the newly created methods,we formed a database that the rate of TGEV positive infect piglets in 1-2weeks was 17%,fatality rate was 100%. TGEV positive infect piglets in 3-4 weeks were 14%,fatality rate was 78. 6%. TGEV positive infect piglets in 5-6 weeks were 9%,fatality rate was 44. 4%. TGEV positive infect piglets in 7-8 weeks were 4%,fatality rate was 25%,TGEV positive infect piglets in 9-10 weeks were 1%,fatality rate was 0%,which showed a downward trend with piglets week-old increasing. In Chengdu,Deyang,Mianyang,Suining,Nanchong region,TGEV positive infect piglets in 1-10 weeks were15%,6%,10%,6%,8% respectively. The results demonstrated in this study illustrate that this was a specific,sensitive and repeatable method which could be effectively utilized in clinical diagnosing the TGEV.

关 键 词：猪传染性胃肠炎病毒(TGEV) N基因 RT-PCR 诊断 

分 类 号：S852.65[农业科学—基础兽医学]