BVDV一步法双重荧光RT-PCR检测方法的建立及应用  

Development and application of a one-step dual real-time RT-PCR for detection and typing of bovine viral diarrhea virus

作　　者：王建昌 王金凤 崔元[2] 刘立兵 袁万哲[2] 

机构地区：[1]河北出入境检验检疫局检验检疫技术中心,河北石家庄050051 [2]河北农业大学动物医学院,河北保定071001

出　　处：《中国兽医学报》2018年第2期245-251,共7页Chinese Journal of Veterinary Science

基　　金：国家质量监督检验检疫总局科技计划资助项目(2015IK093)

摘　　要：为实现对牛病毒性腹泻病毒（bovine viral diarrhea virus，BVDV）的快速检测和分型，本试验根据OenBank已发表的BVDV1和BVDV25’UTR基因特异性保守区域设计特异性引物和探针，以体外转录病毒RNA作为模板，建立了BVDV1和BVDV2一步法双重荧光RT-PCR方法。结果显示，双重荧光RT—PCR对BVDV1和BVDV2的检测下限均为10^2拷贝；具有较强的特异性，对口蹄疫病毒、猪瘟病毒、Hobi样病毒、牛传染性鼻气管炎病毒、施马伦贝格病毒等病毒核酸的扩增均为阴性；BVDV1和BVDV2的扩增均在10^2～10^7拷贝内呈现良好的线性关系，标准曲线的相关系数（R^2）分别为0．999和0．998；方法重复性好，BVDV1和BVDV2的变异系数（CV值）分别在0．68％～1．87％和1.25％～1．900A。本试验对665份样品进行检测，共检出BVDV阳性样品45份，其中BVDV1阳性样品39份，BVDV2阳性样品5份，BVDV1和BvDv2均阳性样品1份。结果表明，本试验建立的BVDV一步法双重荧光RT-PCR方法敏感性高、特异性强、重复性好，可广泛应用于BVDV的快速检测、分型和流行病学调查。To rapidly detect and type the bovine viral diarrhea virus（BVDV）,the universal primers and probes were designed from the specific and conserved regions of 51UTR of the known BVDV1 and BVDV2 in the GenBank. The one-step dual real-time RT-PCR assay was developed using the in vitro transcribed RNA as the templates. The detection limit of the dual real-time RT-PCR was 102copies for BVDV1 and BVDV2. The assay demonstrated its good specificity for BVDV1 and BVDV2, but not cross reaction to foot-and-mouth disease virus（FMDV）, classical swine fever virus （CSFV）, Hobi-like virus, infectious bovine rhinotracheitis （IBRV） and Schmallenberg virus （SBV）. The assay showed the good linear amplification in the range of 102-107copies,with correlation coef- ficient（R2） values of 0. 999 and 0. 998 for BVDV1 and BVDV1, respectively. The assay was highly reproducible with the coefficient variation（CV） values ranging from 0. 68%-1. 87% for BVDV1 and 1.25%-1.90% for BVDV2. By the dual real-time RT-PCR analysis of 665 samples,45 BVDV positive samples were detected,in which 39 samples were BVDV1 positive, 5 samples were BVDV2 positive, and 1 sample was both BVDV1 and BVDV2 positive. The developed BVDV one-step dual real-time PCR was highly sensitive,specific and reproducible,which represented a reliable and effective tool for the rapid de- tection, typing and epidemiology investigation of BVDV.

关 键 词：牛病毒性腹泻病毒 一步法 双重荧光RT—PCR 检测 分型 

分 类 号：S852.65[农业科学—基础兽医学]