兔出血症病毒与欧洲野兔综合征病毒复合RT-PCR检测方法的建立  

Development of a complex RT-PCR assay for the detection and differentiation of rabbit hemorrhagic disease virus and European brown hare syndrome virus

作　　者：王印[1] 杨泽晓[1,2] 韩雪清[3] 汪开毓[1] 姚学萍[1] 白瑜[1] 

机构地区：[1]四川农业大学动物医学院,四川雅安625014 [2]动物疫病与人类健康四川省重点实验室,四川雅安625014 [3]中国检验检疫科学研究院,北京100029

出　　处：《中国兽医科学》2011年第11期1165-1170,共6页Chinese Veterinary Science

基　　金：教育部"长江学者和创新团队发展计划"创新团队项目(IRT0848);四川农业大学双支计划项目(00270401);博士后培养经费项目(04310621);四川省教育厅青年基金项目(11ZB064)

摘　　要：为建立用于兔出血症病毒(RHDV)和欧洲野兔综合征病毒(EBHSV)诊断鉴别的快速检测方法,根据GenBank中的RHDV和EBHSV基因组序列,设计合成5条特异性引物和9条重叠延伸引物,分别以通过重叠延伸PCR人工合成的EBHSV VP60基因中359bp基因片段构建的重组质粒pGM-T-EBHSV和RHDV RT-PCR产物构建的pGM-T-RHDV为模板,经过反应条件的优化、敏感性试验和特异性试验,初步建立了RHDV和EBHSV的复合RT-PCR检测方法,并对20份临床样品和5份RHDV人工感染样品进行了检测。结果表明,建立的复合RT-PCR方法具有良好的特异性和敏感性,可分别扩增出RHDV和EBHSV的特异性目的片段192bp和335bp,对2种病毒的检测限度均可达到50copies的目的基因片段,兔巴氏杆菌、兔大肠杆菌和沙门氏菌核酸扩增均为阴性。样品检测结果显示,5份人工感染样品的RHDV检出率为100%,20份临床样品的RHDV检测为阴性。To develop a rapid method for the detection and differentiation of European brown hare syndrome virus（EBHSV） and rabbit hemorrhagic disease virus（RHDV）,5 specific primers and 9 overlap extention primers were designed to amplify a 335bp EBHSV specific DNA fragment and a 192bp RHDV specific DNA fragment according to the capsid protein（VP60） gene of EBHSV and RHDV in GenBank by using overlap extension PCR and reverse transcriptase polymerase chain reaction（RT-PCR） respectively. Then two recombinant plasmids named pGM-T-EBHSV and pGM-T-RHDV were constructed. The complex RT-PCR method for rapid detection and differentiation of EBHSV and RHDV was developed and optimized with the sensitivity and specificity evaluated with the two constructed recombinant plasmids and other main pathogens. Results showed good specificity and sensitivity of the complex RT-PCR method to EBHSV and RHDV with the sensitivity reached about 50 copies of cloned viral genomic fragments of EBHSV and RHDV,and no cross-reaction with other virus such as Pasteurella multocida,Escherichia coli and Salmonella from rabbits. Furthermore,the complex RT-PCR method was performed for the detection of RHDV in 20 clinical samples and 5 experimentally infected samples with the results of 0 and 100%,respectively,which indicated a practical usage of the method.

关 键 词：兔出血症病毒 欧洲野兔综合征病毒 反转录-聚合酶链反应 重叠延伸PCR 

分 类 号：S852.659.6[农业科学—基础兽医学]