BTV-2型3型4型7型12型HPCE检测方法的建立  

Establishment of HPCE for the simultaneous identification and detection of bluetongue virus 2,3,4,7 and 12

作　　者：高源 黄秋华 聂福平 杨俊 王昱 艾军 唐昌杰 史梅梅 李贤良 王国民 胡世君[1] GAO Yuan;HUANG Qiu-hua;NIE Fu-ping;YANG Jun;WANG Yu;AI Jun;TANG Chang-jie;SHI Mei-mei;LI Xian-liang;WANG Guo-min;HU Shi-jun(College of Animal Science,Southwest University,Chongqing 402460,China;The Technology Center of Chongqing Customs/Engineering Center for the Research of Imported Terrestrial Animal Disease Prevention and Control in Chongqing/State Key Laboratory of Bovine Diseases,Chongqing 400020,China;The Technology Center of Yunnan Customs China,Kunming 650228,China)

机构地区：[1]西南大学动物科学学院,重庆荣昌402460 [2]重庆海关技术中心重庆市进境陆生动物疫病防控技术研究工程中心国家牛病检测重点实验室,重庆江北400020 [3]云南海关技术中心,云南昆明650228

出　　处：《中国兽医科学》2019年第11期1361-1367,共7页Chinese Veterinary Science

基　　金：重庆市重大应用开发项目（cstc,2015yykfC80001）;质检公益性行业科研专项（2013100093）

摘　　要：为了建立同时鉴别检测蓝舌病毒(BTV)2型、3型、4型、7型及12型的快速方法,针对BTV不同血清型的VP2基因保守区,采用Ge XP原理,设计了5对特异性嵌合引物和1对通用引物,优化建立了可单管同步鉴别BTV2型、3型、4型、7型及12型的HPCE方法。该方法仅对BTV 2型、3型、4型、7型、12型的病毒核酸有扩增,对BTV的其他基因型以及小反刍兽疫病毒、口蹄疫病毒均无扩增,特异性好。敏感性试验结果显示:该方法对单一病毒核酸的最低检测量为100~1 000 copies/μL;对五种血清型病毒混合核酸的最低检测量为100 copies/μL。组内重复试验与组间重复试验结果均一致,重复性好。临床样品和模拟样品检测结果显示,本研究建立的HPCE方法与OIE推荐的套式RT-PCR敏感性一致,且该方法可以特异、敏感、快速地鉴别蓝舌病毒(BTV)2型、3型、4型、7型及12型。A rapid and high-throughput method was developed for simultaneous detection and differentiation of bluetongue virus(BTV) serotypes(2,3,4,7 and 12) by multiplex PCR based on a Genome Lab Gene Expression Profiler(GeXP) analyser.Five pairs of chimeric primers consisting of both the genespecific primer and a universal primer were designed and used for amplification.With condition optimization,a single tube multiplex RT-PCR method for simultaneous identification of BTV serotype 2/3/4/7/12 was established in this study.The results showed that the detection method can specifically amplify BTV-2/3/4/7/12,without amplification of the nucleic acid of other BTV serotypes,Peste des Petits Ruminants virus,and foot-and-mouth disease virus,which indicated strong specificity.The sensitivity test results show that this assay achieved a sensitivity of 100-1000 copies/μL for a single virus and 100 copies/μL when all of the five pre-mixed viral targets were present.Furthermore,The assay developed in this study is highly sensitive and presents low intra-and inter-run variation and it was 100% specific when 100 clinical samples were tested in comparison with the OIE nested RT-PCR method.In conclusion,the HPCE assay is a rapid,cost-effective,sensitive,specific and high throughput method for simultaneously detecting bluetongue virus(BTV) serotypes(2,3,4,7 and 12).

关 键 词：蓝舌病毒 多重RT-PCR 血清型 鉴别 

分 类 号：S852.659.4[农业科学—基础兽医学]