牛肠道病毒TaqMan荧光定量RT-PCR检测方法的建立  

Establishment of real-time RT-PCR method for detection of bovine enterovirus

作　　者：吴丹[1,2] 吴涛[3] 张鹤晓[2] 高志强[2] 蒲静[2] 张伟[2] 乔彩霞[2] 谷强[2] 夏春[1] 

机构地区：[1]中国农业大学动物医学院,北京100193 [2]北京出入境检验检疫局,北京100026 [3]中国兽医药品监察所,北京100081

出　　处：《中国预防兽医学报》2012年第11期903-906,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家质检总局科研项目"牛肠道病毒快速检测技术研究"(2007IK021)

摘　　要：为建立快速、准确地检测牛肠道病毒(BEV)及对北京周边地区牛群中BEV感染情况进行检测,本研究通过设计合成扩增BEV 5'UTR序列的引物及探针建立了检测BEV的通用型TaqMan荧光定量RT-PCR快速检测方法并对反应条件进行了优化。该方法对BEV的最小检测量为0.1 TCID50/0.1 mL,敏感性比病毒分离法高10倍。而且该方法对其他牛相关病毒均无交叉反应。用3批试剂对3个稀释度的BEV培养物进行批内和批间重复性试验,其变异系数均小于1.1%,表明该方法具有良好的重复性。采用该方法对北京周边地区牛群采集的852份临床样品进行检测,并与病毒分离方法比较,两种方法检测结果完全一致。因此,本研究所建立的BEVTaqMan RT-PCR检测方法具有快速、准确、特异性强、敏感性高、重复性好的优点,为国内BEV的检测提供了有力的技术支持。本研究首次通过病毒核酸检测证明了我国部分地区的牛群中存在BEV感染。Bovine enterovirus （BEV） is newly emerged in China. To establish a rapid method for detection of BEV infections, a TaqMan based real-time RT-PCR was developed with a pair of primer for 5＇UTR sequence and a probe of BEV. Under the optimized conditions, the TaqMan RT-PCR assay was specifically detection of BEV and no cross-reactions with other related bovine viruses. In addition, the detection limit of this method was 0.1 TCID~/0.1 mL of BEV which was 10 times sensitivity than the virus isolation and the variation coefficients of intra- and inter-assay were around 1.1%. Furthermore, tested on 852 clinical samples collected from Beijing areas, the results showed that 132 samples were positive, which were completely consistent with the results by virus isolation. Our data showed that the TaqMan RT-PCR assay established in the study was rapid, high specificity, high sensitivity and repeatability, which provides a useful technical support for detection of BEV detection.

关 键 词：牛肠道病毒 TAQMAN RT-PCR 病毒核酸检测 

分 类 号：S852.65[农业科学—基础兽医学]