兔出血症病毒与兔出血症病毒2型复合RT-PCR检测方法的初步研究  

Development of a complex RT-PCR for the detection and differentiation of rabbit hemorrhagic disease virus and rabbit hemorrhagic disease virus 2

作　　者：杨泽晓[1] 赵希仑 李岩[2] 姚学萍[1] 王印[1,3] 刘亚东[1] 蒙正群 耿毅[1] 白瑜[1] 

机构地区：[1]四川农业大学动物医学院,四川成都611130 [2]四川农业大学动物科技学院,四川成都611130 [3]动物疫病与人类健康四川省重点实验室,四川成都611130

出　　处：《中国兽医科学》2016年第6期710-716,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31402222);教育部"长江学者和创新团队发展计划"创新团队项目(IRTO848);四川省科技支撑计划项目(2016NZ0002);四川农业大学双支计划项目(00270401)

摘　　要：为建立用于兔出血症病毒(RHDV)与兔出血症病毒2型(RHDV2)鉴别诊断的检测方法。根据Gen Bank已公布的RHDV和RHDV2的VP60基因,设计合成6对特异性引物和10条搭桥引物,采用RT-PCR和搭桥PCR技术分别获取RHDV和RHDV2的VP60基因中保守区片段,构建重组质粒p MD-19T-RHDV2、p MD-19T-RHDV441和p MD-19T-RHDV294,并以其为模板,经过反应条件优化、敏感性试验、特异性试验以及人工感染RHDV样品和临床样品的应用,对RHDV与RHDV2的复合RT-PCR检测方法进行了初步研究。结果显示,建立的RHDV与RHDV2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RHDV2的VP60保守区基因片段大小为294 bp和435 bp,RHDV与RHDV2的检测限度分别为160和230个拷贝的靶基因片段,而p GM-T-EBHSV(已构建)、兔巴氏杆菌、兔源大肠杆菌和沙门菌等病原的检测结果均为阴性。样品检测结果显示,人工感染样品的RHDV检出率为100%(5/5),30份临床检样RHDV和RHDV2检测结果均为阴性。To develop a rapid method for the detection and differentiation of rabbit hemorrhagic disease virus（RHDV）and rabbit hemorrhagic disease virus 2（RHDV2）,6 specific primers and 10 overlapping oligo primers were designed according to the VP60 gene sequences of RHDV and RHDV2 published in Gen Bank.A 435 bp DNA fragment of the RHDV2 capsid protein（VP60） gene was synthesized in vitro by using overlap extension PCR.Two DNA fragments about 441 bp and 294 bp of the RHDV capsid VP60 gene wereamplified by using reverse transcriptase polymerase chain reaction（RT-PCR） to construct the recombinant plasmids p MD-19T-RHDV2,p MD-19T-RHDV441 and p MD-19T-RHDV294,respectively Then a complex RT-PCR assay for rapid detection and differentiation of RHDV and RHDV2 was developed and optimized after a series of tests,including,optimization of reaction conditions,the sensitivity and specificity tests,and the application tests of 35 samples.The results showed the established complex RT-PCR method for the detection of RHDV and RHDV2 amplified the specific products size of RHDV and RHDV2 were separately 294 bp and 435 bp,and it had a good specificity and sensitivity.The complex RT-PCR detection limits of RHDV and RHDV2 were separately 160 copies and 230 copies of the cloned viral genomic fragments,and no amplification for p GM-T-EBHSV,Pasteurella multocida,E.coli and Salmonella from rabbits detection by this approach.And the application tests showed the positive rate of the experimentally infected samples was 100%,and there were no RHDV or RHDV2 detection in the tested clinical samples.

关 键 词：兔出血症病毒 兔出血症病毒2型 RT-PCR 搭桥PCR 

分 类 号：S855.3[农业科学—临床兽医学]