牛传染性鼻气管炎病毒内标荧光定量PCR检测方法的建立与应用  

Establishment and application of fluorescent quantitative real-time PCR with internal amplification control for the detection of infectious bovine rhinotracheitisv virus

作　　者：季新成[1,2] 牛国辉[3] 员丽娟[2] 张彦明[1] 李鹏[2] 段晓东[2] 于学辉[2] 

机构地区：[1]西北农林科技大学,陕西杨凌712100 [2]新疆出入境检验检疫局,新疆乌鲁木齐83006 [3]新疆大学,新疆乌鲁木齐830000

出　　处：《中国兽医学报》2010年第6期738-743,747,共7页Chinese Journal of Veterinary Science

基　　金：国家质量监督检验检疫总局资助项目(2007IK023)

摘　　要：根据牛疱疹病毒1型(BHV1)gB基因序列,设计高度保守的引物和荧光探针,并扩增包含有荧光PCR扩增区域的核酸片段,通过凝胶纯化回收,将该片段克隆至pMD18-T载体,经鉴定后获得目标模板。通过碱基重排和引物设计,用搭桥法PCR扩增,获得BHV1荧光定量PCR内标模板。对内标模板的添加量和反应条件进行优化,建立了含有内标物的荧光定量PCR检测体系。该体系可以检测到10个拷贝/PCR反应的重组质粒,对BHV1可检测到0.01TCID50的病毒粒子,灵敏度比常规PCR和病毒分离高10～100倍,与不加内标的荧光PCR检测灵敏度相当。更重要的是,内标模板可以对反应体系进行监测,指示并校正假阴性结果,从而达到提高PCR检测准确率的目的。对临床收集的40份牛精液(其中8份已经鉴定为阳性)和10份牛鼻腔拭子(其中2份已经鉴定为阳性)用该体系进行检测,均得到预期结果。该方法的建立可用来对临床样品中BHV1的快速检测和实验室的质量控制。According to the gB gene sequence of bovine herpesvirus 1 （BHV1）,specific primers and fluorescent probe were designed to amplify the nucleotide fragment containing the fluorescent quantitative real-time PCR（FQ-PCR） amplify region.Cloned the fragment into pMD18-T vector and got the target template.The internal amplification control （IAC） template of FQ-PCR was achieved by nucleic acid sequenced and amplifying with bridge-building PCR.By optimizing the quantity of the IAC template and the reaction condition,the FQ-PCR detection system with IAC was established.The detective limit was about 10 copies plasmids and 0.01TCID50 BHV1 virus each PCR reaction.Compared with conventional PCR and virus separation method,the FQ-PCR with IAC was 10 to 100 folds more sensitive.This result was almost coincident with FQ-PCR without IAC.The detection results from clinical samples indicated that the FQ-PCR was a rapid,sensitive,specific and reproducible.What is more,the IAC in the real-time PCR system can indicate and proofread false negative results.Using this method,40 bovine semen （8 were identified for positive） and 10 nasal swabs （2 were identified for positive） were detected,3 semen samples showed the present of inhibitor.After a further purification to DNA,all the samples got the expected results.It suggested that this method was useful for the rapid detection of BHV1 and lab quality control.

关 键 词：牛传染性鼻气管炎病毒 荧光定量PCR 内标 共扩增 

分 类 号：S852.65[农业科学—基础兽医学]