牛病毒性腹泻病毒1型和2型双重RT-PCR检测方法的建立  

Establishment of Duplex RT-PCR for Detection of BVDV1 and BVDV2

作　　者：王克栋[1,2] 熊浩[1,2] 季新成[2] 冉多良[1] 彭武丽[2] 于学辉[2] 史茜[2] 

机构地区：[1]新疆农业大学动物医学学院,新疆乌鲁木齐830052 [2]新疆出入境检验检疫局,新疆乌鲁木齐830063

出　　处：《动物医学进展》2013年第10期21-25,共5页Progress In Veterinary Medicine

基　　金：新疆自治区高新技术项目(201010101;201141147);国家质量监督检验检疫总局科研项目(2011IK017)

摘　　要：根据GenBank已发表的牛病毒性腹泻病毒(BVDV)基因1型和2型毒株的5′端非编码区(5′-UTR)保守序列的差异,设计2对特异性引物,并以牛GAPDH基因为内标,设计1对特异性引物,建立牛病毒性腹泻病毒分型与内标三重RT-PCR检测方法。3对引物可分别扩增出的片段大小为127、94、152bp,与预期大小相符。经反应条件优化,该方法的灵敏度为10°TCID50,与不加内标检测灵敏度相当。经临床样品检测验证,该方法具有较好的特异性和重复性,可用来对牛病毒性腹泻病毒分型进行快速准确的检测。According to the difference of the 5′-untranslated region （5r-UTR） between BVDV1 and BVDV2 in GenBank, two pairs of primers were designed, and making the bovine GAPDH gene as the internal amplification control, a specific pair of primers were also designed in order to establishing an internal amplification control triplex RT-PCR detection method for classifying bovine viral diarrhea virus. These three primers can amplify fragments of 127 bp, 94 bp, and 152 bp respectively, the results was consistent with the theory. Through the optimization of reaction conditions, the sensitivity of this method was 100 TCID80, it was same with that without internal amplification control. The clinical sample tests results showed the method had a higher sensitivity and reliability, so it can be used to classify BVDV1 and BVDV2 rapidly and accurately.

关 键 词：牛病毒性腹泻病毒 GAPDH基因 基因分型 内标 双重RT-PCR 

分 类 号：S852.659.6[农业科学—基础兽医学]