牛病毒性腹泻病毒内标双重TaqMan荧光RT-PCR方法的建立及初步应用  

Establishment and application of the duplex TaqMan real-time RT-PCR with internal control for bovine viral diarrhea virus detection

作　　者：季新成[1] 史茜[1] 郭春娟[2] 王科珂 冉多良[2] 

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

出　　处：《中国预防兽医学报》2014年第8期646-650,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：新疆自治区高技术研究发展项目(201010101201141147);国家质量监督检验检疫总局科研项目(2011IK011;2011IK017)

摘　　要：为建立牛病毒性腹泻病毒（BVDV）内标双重TaqMan荧光RT-PCR方法,本研究根据BVDV 5＇-UTR区序列设计引物和荧光标记探针,通过重叠延伸PCR（SOE-PCR）扩增获得内标模板,经体外转录得到可以作为内标物的cRNA。经反应条件的优化,建立了BVDV内标双重TaqMan荧光RT-PCR检测体系。结果表明：当cRNA浓度为103拷贝/μL时,二者均有较好的扩增曲线;该方法对I型BVDV的检测灵敏度为0.25 TCID50,对II型BVDV的检测灵敏度为2.5 TCID50;利用该方法对猪瘟病毒等其他相关核酸的检测结果均为阴性;重复性检测Ct值变异系数为0.54~4.73。对506份临床样品检测,结果有23份样品为阳性,阳性样品检出数量和编号与不含内标的单一荧光RT-PCR方法完全一致,表明该方法可以用来对BVDV进行准确、快速检测,并且可以对检测结果进行质量监控。In order to establish a duplex TaqMan real-time RT-PCR method with internal control （IC） for bovine viral diarrhea virus （BVDV） detection, specific primers and fluorescent probes were designed according to the published sequence of BVDV 5＇-UTR. IC （cRNA） template of the real-time RT-PCR was amplified by gene splicing overlap extension PCR （SOE-PCR） and then prepared by transcription in vitro. After optimizing the quantity of the IC template and the reaction condition, the optimum concentration of cRNA was 103 copies/μL. The sensitivity of the assay was 0.25 TCID50 and 2.5 TCID50 for BVDV I and II detection, respectively. The reproducible detection indicated the Ct value of the coefficient of variation was 0.54 to 4.73. A total of 506 clinial samples were test by this method, and 23 samples were positive which was coincident with single real-time PCR. This study indicated that this method could be used for rapidly BVDV detection.

关 键 词：牛病毒性腹泻病毒 内标 共扩增 TaqMan荧光RT-PCR 

分 类 号：S852.65[农业科学—基础兽医学]