摘要
马病毒性动脉炎(Equine viral arteritis,EVA)是由马动脉炎病毒引起的一种通过呼吸道或生殖器官传播的传染病,是危害养马业的重大疾病之一,被世界动物卫生组织(OIE)列入检疫名录。近年来,我国社会经济不断发展和人们生活水平不断提高,养马和赛马越来越多,马术比赛国际化交流日益增多,该病的传播风险也越来越高。马病毒性动脉炎国际贸易指定诊断方法为病毒分离(仅精液)和中和试验,这两种诊断方法检测技术要求高、检测周期长,不利于对该病的快速应对和防控。为防止该病的传入,有必要建立和应用快速、高效、操作简便的诊断方法。本研究分别针对马病毒性动脉炎的抗体和病原建立了两种快速诊断方法,弥补了国际贸易指定方法的缺点,为该病的防控提供了技术支撑。
1、间接ELISA抗体检测方法应用RT-PCR技术,以马动脉炎病毒基因组模板扩增出马动脉炎病毒N基因,将N基因插入到pET-32a原核表达获得约30kD的N蛋白,Western-blotting结果显示N蛋白具有良好抗原性,通过镍柱纯化获得N蛋白,在此基础上建立了马动脉炎病毒抗体间接ELISA诊断方法,制订了标准操作程序。
应用建立的问接ELSIA诊断方法对2010年至2012年来自吉林、北京、天津、广东等6个不同地区疑似马病毒性动脉炎血清557份进行抗体检测,检出马动脉炎病毒抗体阳性血清11份,与病毒中和抗体检测结果完全相符。
2、RT-LAMP病毒检测方法针对马动脉炎病毒的膜基质蛋白M基因保守序列,设计特异性引物,优化反应条件,建立了马动脉炎病毒的RT-LAMP诊断方法。
2010年至2012年应用所建立的马动脉炎病毒RT-LAMP检测方法,在新疆、辽宁、黑龙江、内蒙古等4个省进行应用,2年时间检测精液、全血等样品1826份,共计检测出阳性9头份,对阳性马及时采取隔离和扑杀等措施,防止了马动脉炎病的传入境内。
该方法已经制订成宁波局标准。研发的马动脉炎病毒的RT-LAMP诊断试剂盒专利正在申报中(已经受理,申请号：201210056298.9)
通过对马动脉炎病毒的两种快速诊断方法的研究,为应对未来突发性检疫任务做好了技术储备。
Equine viral arteritis (EVA) is one of the most important disease included in the quarantine disease List by OIE and which is a contagious viral disease transmitted through respiratory passages and direct sexual contact caused by Equine arteritis virus (EAV). With the rapid development of community economy and the constant improvement of living standard in our country, more and more people like horse raising, horseback riding and horse racing. The risk of the EAV spreading became more and more serious because of the increasing of international equestrian competition. Virus isolation (only semen) and neutralization test were specified as the prescribed methods for EVA diagnosis during international trade. But those methods require higher detection technology and longer detection cycle which is harmful to the prevention and control of the disease. Therefore, a fast, efficient, and simple diagnostic method must be established to prevent the spread of the disease to China. This study were developed two rapid diagnostic methods according to the antibodies and pathogens of the EVA which compensate for the shortcomings of the specified method of international trade and provide technical support for the prevention and control of EVA.
1. Indirect ELISA antibody detection method:The N gene of the EAV was amplified by RT-PCR using specific primers designed according to the sequence of N gene in GenBank. And then it was cloned into pET-30a(+) and expressed in E. coli BL21(DE3). Western blot analysis showed that the recombinant protein reacted specifically with EAV positive serum. An indirect ELIS A was developed to detect EAV antibody using the purified recombinant N protein as coating antigen. The assay was specific, sensitive and reproducible, which could be used as a rapid serology detection method for monitoring the EAV antibody and epidemiologic survey of EAV.
557suspected EVA sera samples between2010and2012from Jiling, Beijing, Tianjin, Heilongjiang, the Inner Mengolia and Guangdong areas were detected by this assay. The results showed that about11samples were positive which verified well with the virus neutralization test result.
2. RT-LAMP virus detection method:The reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was established using a set of specific primers designed and synthesized according to the membrane protein M gene sequence of EAV.
The RT-LAMP assay was used to detect the semen and whole blood samples from Xinjiang, Liaoning, Heilongjiang and the Inner Mengolia between2010and2012.9positive were checked out from1826samples. To preventing the spread of EVA, positive horses were isolated and slaughtered timely.
The method has been developed into Ningbo Bureau of standards. The patents of RT-LAMP diagnostic kits for EVA are under applied (the application number:201210056298.9).
According to the research on the two rapid diagnostic methods for EVA, a good technical preparation was set for the precipitate task of quarantine of EVA for future.
引文
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