牛轮状病毒VP7基因荧光定量RT-PCR检测方法的建立  

Establishment of real-time PCR for detecting VP7 gene of bovine rotavirus

作　　者：魏锁成[1] 巩转娣[2] 车团结 田风林[1] 

机构地区：[1]西北民族大学生命科学与工程学院,甘肃兰州7300030 [2]西北民族大学医学院附属医院,甘肃兰州730030 [3]兰州百源基因技术有限公司,甘肃兰州730000

出　　处：《中国预防兽医学报》2013年第2期151-154,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：2007年甘肃省科技支撑计划项目(0708NKCA079);甘肃省农业生物技术研究与应用开发项目(GNSW-2010-10)

摘　　要：为建立快速特异的定量检测牛轮状病毒(BRV)VP7基因的荧光定量PCR检测方法,本研究设计扩增BRV VP7基因的特异性引物,将扩增的VP7基因片段(342 bp)克隆于pMD18-T载体中(pMD-VP7),作为重组质粒标准品,建立EvaGreen荧光定量RT-PCR(qRT-PCR)检测方法。经反应条件优化,结果表明该方法的最低检测量为8.03拷贝/μL。该方法对牛病毒性腹泻病毒、牛冠状病毒、猪流行性腹泻病毒及牛结核分枝杆菌的检测结果均为阴性,表明其具有良好的特异性。批内和批间重复变异系数均小于3%。采用该方法对12份ELISA阳性样本检测出10份阳性,符合率83.33%。本研究建立的BVR VP7 qRT-PCR检测方法具有特异、灵敏、快速和对标本的检测能力较强等特点,可以用于临床诊断和流行病学调查。In the present study, an EvaGreen real-time RT-PCR （qPCR） was developed for detection of bovine rotavirus （BRV） with the specific primers designed according to VP7 gene of BRV in GenBank. The 342 bp VP7 gene fragment of BRV was amplified by RT-PCR from total RNA extracted from BRV strain AV-51 and cloned into the pMD18-T vector （pMD-VP7）, as the standard of recombinant plasmid. Under the optimized reaction conditions, the detecting limit of qRT-PCR was 8.03 copies/μL and no cross reactions with the bovine viral diarrhea virus, bovine coronavirus, porcine epidemic diarrhea virus and bovine Mycobacterium tuberculosis. In addition, the coefficients of variation in both intra- and inter-assay were less than 3%. The coincidence rate to ELISA method was 83.33%. The established qRT-PCR in this study was specificity, sensitivity and reproducibility. It could be applied to clinical diagnosis and epidemiological survey of BRV.

关 键 词：轮状病毒 VP7基因 实时荧光定量PCR 

分 类 号：S852.65[农业科学—基础兽医学]