牛病毒性腹泻病毒、牛肠道病毒、牛轮状病毒、牛冠状病毒四重一步法RT-PCR鉴别检测方法的建立与应用  

Development and Application of Quadruple One Step RT-PCR Method for Identifi cation of Bovine Viral Diarrhea Virus,Bovine Enterovirus,Bovine Rotavirus and Bovine Coronavirus

作　　者：季彬[1,2] 彭轶楠 叶泽[1,2] 王莎莎 王治业 JI Bin;PENG Yinan;YE Ze;WANG Shasha;WANG Zhiye(Institute of Biology,Gansu Academy of Sciences,Gansu 730030,China;Key Laboratory of Microbial Resources Exploition Application,Lanzhou 730030,China;Lanzhou Second People's Hospital,Lanzhou 73003,China)

机构地区：[1]甘肃省科学院生物研究所,兰州730030 [2]甘肃省微生物资源开发利用重点实验室,兰州730030 [3]兰州市第二人民医院,兰州730030

出　　处：《中国动物传染病学报》2023年第5期120-127,共8页Chinese Journal of Animal Infectious Diseases

基　　金：甘肃省科技计划项目(20YF8FA128)。

摘　　要：为了建立一种能够同时、快速鉴别检测牛病毒性腹泻病毒(BVDV)、牛肠道病毒(BEV)、牛轮状病毒(BRV)、牛冠状病毒(BCV)的方法,根据BVDV 5'UTR基因、BEV 5'UTR基因、BRV VP6基因、BCV N基因设计引物,建立了能够同时鉴别检测BVDV、BEV、BRV、BCV的四重一步法RT-PCR方法,该方法检测IBRV、BPIV-3、BRSV均为阴性,对BVDV、BEV、BRV、BCV的核酸检测下线分别为3.28、28.2、23.6、25.0 pg,与现有标准或商品化RT-PCR试剂盒的符合率均为100%,批内和批间重复性试验的检测结果均完全一致,检测222份临床样品的BVDV、BEV、BRV、BCV阳性率分别为15.32%、18.02%、6.31%和4.05%。说明四重一步法RT-PCR方法特异性好、灵敏度高、稳定性强,可用于BVDV、BEV、BRV、BCV的临床鉴别诊断、流行病学调查以及疫病防控。To develop a rapid and simple identification method of Bovine viral diarrhea virus(BVDV),Bovine enterovirus(BEV),Bovine rotavirus(BRV)and Bovine coronavirus(BCV),the specific primers were designed based on the sequences of BVDV 5'UTR gene,BEV 5'UTR gene,BRV VP6 gene and BCV N gene in GenBank for development of a quadruple one-step RT-PCR.This RT-PCR method was negative to IBRV,BPIV-3 and BRSV.The detection limits of this RT-PCR method were 3.28 pg for BVDV,28.2 pg for BEV,23.6 pg for BRV and 25.0 pg for BCV.The test agreement of this RT-PCR with the existing standard or commercial RT-PCR kit was 100%.In addition,the intra and inter batch repeatability tests were completely consistent.Then,this RT-PCR method was used to test 222 clinical samples.The results showed that the positive rates of these clinical samples were 15.32%for BVDV,18.02%for BEV,6.31%for BRV and 4.05%for BCV.In conclusion,the quadruple one-step RT-PCR method developed here had good specificity,high sensitivity and strong stability,and could be used for clinical differential diagnosis,epidemiological investigation and epidemic prevention and control of BVDV,BEV,BRV and BCV.

关 键 词：牛病毒性腹泻病毒 牛肠道病毒 牛轮状病毒 牛冠状病毒 一步法RT-PCR 

分 类 号：S858.23[农业科学—临床兽医学]