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.
出处
《中国兽医科学》
CAS
CSCD
北大核心
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)
