牛轮状病毒实时荧光定量PCR检测方法的建立  

Development of real-time PCR for detection of bovine rotavirus

作　　者：王梓[1] 刘佳丽[1] 赵莉[1] 王瑞翀[2] 李一经[1] 王丽[1] 唐丽杰[1] 徐义刚[1] 刘敏[1] 乔薪瑗[1] 

机构地区：[1]东北农业大学动物医学学院,黑龙江哈尔滨150030 [2]黑龙江省疾病控制中心,黑龙江哈尔滨150030

出　　处：《中国兽医科学》2017年第6期750-754,共5页Chinese Veterinary Science

基　　金："十三五"国家重点研发计划项目(2016YFD0500904)

摘　　要：为建立快速特异的定量检测牛轮状病毒的荧光定量PCR方法,本研究从牛轮状病毒NCDV株DNA中扩增的VP6基因片段(211 bp)并克隆于pMD19-T载体(p MD19-T-VP6)作为重组质粒标准品,建立了SYBR Green I荧光定量PCR检测方法。结果表明,VP6片段长211 bp,经鉴定所构建的重组质粒成功;用该质粒测得real-time PCR的最低检测量为15.39 copies/L。用该方法检测猪传染性胃肠炎病毒、牛细小病毒、猪流行性腹泻病毒及伪狂犬病病毒的结果均为阴性,表明其具有良好的特异性。重复性试验结果表明,批内和批间重复变异系数均小于3%,表明该方法具有良好的重复性。以上结果表明,本研究建立的基于VP6基因的牛轮状病毒qRT-PCR检测方法具有特异、灵敏、快速等特点,可用于牛轮状病毒的早期诊断。To establish a rapid and specific real-time fluorescent quantitative PCR assay to detect bovine rotavirus,the VP6 gene fragment of bovine rotavirus NCDV strain（211 bp） was cloned into the pMD19-T vector as the standard of recombinant plasmid and a SYBR Green I fluorescent quantitative PCR detection method was established. The results showed that the recombinant p]asmid containing 211 bp length VP6 gene was successfully constructed and the minimum detectable amount of this method was 15.39 copies/~ L. The results for detection of TGEV,BPV,PEDV and PRV by this method were negative,which showed that this method had good specificity. The resu]ts of repetition experiment showed the coefficient of variation of intra-assay and inter-assay were less than 3%,which showed that this method had good repeatability. In the present study,the qRT-PCR detection method basing on VP6 gene of bovine rotavirus was successfully established. This method is specific,sensitiveand rapid,and can be used for the early diagnosis of bovine rotavirus.

关 键 词：牛轮状病毒 VP6基因 实时荧光定量PCR 

分 类 号：S852.659.4[农业科学—基础兽医学]