牛白血病病毒前病毒DNA荧光PCR检测方法的建立及应用  

Application and development of a real-time PCR assay for the detection of bovine leukemia virus provirus DNA

作　　者：王建华 王玉玲 张俊哲 赵丹 肖妍 王乃福 陈本龙 董志珍 WANG Jian-hua;WANG Yu-ling;ZHANG Jun-zhe;ZHAO Dan;XIAO Yan;WANG Nai-fu;CHEN Ben-long;DONG Zhi-zhen(The Animals and Plants and Food inspection Center of the Tianjin Customs,Tianjin 300456,China)

机构地区：[1]天津海关动植物与食品检测中心,天津300456

出　　处：《中国兽医科学》2019年第5期598-603,共6页Chinese Veterinary Science

基　　金：天津海关科技计划项目(TK001-2017)

摘　　要：为快速检测牛外周血淋巴细胞中的牛白血病病毒(bovine leukem ia vir,uBsLV)前病毒DNA,选取BLVenv基因的保守序列设计特异性引物和TaqMan-MGB探针,通过优化反应条件建立检测牛白血病前病毒DNA的荧光PCR方法,并对该方法进行敏感性、特异性和重复性评价及实际样本的验证检测。结果显示,该方法具有很高的特异性和重复性,最低检出限为每个反应可检出3个拷贝数的阳性标准质粒对照。在175头BLV抗体阳性牛中,用该方法共检出127头BLV感染牛。在262头BLV抗体阴性牛中,用该方法检出2头BLV感染牛,该方法与套式PCR之间检测结果的符合率为100%。上述结果表明,本研究建立的方法可用于BLV感染牛的确证。To rapidly detect bovine leukemia virus(BLV) provirus DNA in peripheral blood mononuclear cells(PBMC) from bovine,a real-time PCR targeting the conserved BLV env gene sequences was developed. Furthermore,specificity,repeatability and sensitivity of the assay were evaluated,and performance of the assay was validated by PBMC samples from 437 imported dairy cattle. The assay showed high specificity and reproducibility,and the lower detection limit is 3 copies of the positive standard plasmid control per reaction.Among 175 PBMC samples from the BLV-seropositive bovine,127 were positive by the env real-time PCR.In addition,among 262 PBMC samples from the BLV-seronegative bovine,2 were positive by the env real-time PCR.The assay showed 100% agreement with the nest PCR.In conclusion,the assay developed in this research can be used for confirmation of BLV-infected bovine.

关 键 词：牛白血病 牛白血病病毒前病毒DNA ENV基因 实时荧光PCR方法 

分 类 号：S852.659.6[农业科学—基础兽医学]