Development And Application Of Two Rapid Diagnosis Methods Of Equine Viral Arteritis

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.