兔出血症病毒2型的VP60基因保守区人工合成及RT-PCR检测方法初探  

Artificial synthesis of VP60 conserved gene fragment of rabbit hemorrhagic disease virus type 2 and preliminary study of its RT-PCR detection method

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

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

出　　处：《浙江农业学报》2016年第10期1650-1656,共7页Acta Agriculturae Zhejiangensis

基　　金：国家自然科学基金项目(31402222);"十二五"国家科技支撑计划(2013BAD12B04);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;四川省科技支撑计划(2016N20002)

摘　　要：为建立用于兔出血症病毒2型(RHDV2)的快速检测方法,根据Gen Bank已公布的RHDV和RHDV2VP60基因,设计合成2对特异性引物和10条末端重叠引物,通过重叠PCR人工合成RHDV2 VP60基因中保守区片段,构建重组质粒,并以其为模板,经过反应条件优化、敏感性试验、特异性试验和35份样品检测应用,初步建立RHDV2 RT-PCR检测方法。实验成功合成RHDV2 VP60基因的435 bp保守片段并构建了p MD-19T-RHDV2重组质粒,初步建立的RHDV2 RT-PCR方法具有良好的特异性和敏感性,RHDV2的检测限度可达到230个拷贝的靶基因片段,检测RHDV、p GM-T-EBHSV(已构建)、兔巴氏杆菌、兔源大肠埃希菌和沙门氏菌均无特异性扩增,样品检测结果显示样品中RHDV2核酸阴性。该研究为中国及时进行RHDV2的防控提供技术储备。In order to develop a rapid detection method for RHDV2,4 specific primers and 10 overlapping oligo primers were designed to amplify the conserved RHDV2 VP60 gene specific DNA fragment according to the genome sequences of RHDV and RHDV2 published in Gen Bank. Based on the synthesis of a conserved part of RHDV2 sequence using overlap extension PCR and the construction of recombinant plasmid,RT-PCR assay was firstly established and evaluated in China after a series of tests,including optimization of reaction conditions,sensitivity and specificity tests,and the application tests of 35 samples. It was shown that a 435 bp specific DNA fragment of theRHDV2 capsid protein（ VP60） gene was synthesized in vitro and the recombinant plasmid p MD-19T-RHDV2 was constructed. The sensitivity of the established RT-PCR detection method for RHDV2 could reach about 230 copies of cloned viral genomic fragments of RHDV2,and there was no amplification for RHDV,p GM-T-EBHSV,Pasteurella multocida,Escherichia coli and Salmonella from rabbits by this method. Besides,the application results showed that there were not RHDV2 in the experimental infection RHDV samples and clinical samples. The present research set basis for RHDV2 control study and supplied a back-up method for the rapid detection and prevention of RHDV2.

关 键 词：兔病毒性出血症 RHDV2 RT-PCR 重叠PCR 

分 类 号：S855.3[农业科学—临床兽医学] S829.1[农业科学—兽医学]