Establishment Of Detection Method For Porcine Japanese Encephalitis Virus

This research developed RT-PCR for detection porcine Japanese Encephalitis virus . At present, the detection for porcine Japanese encephalitis virus was only depended on the traditional method: neutralization test, ELISA, HI, but not rely on RT-PCR. The homology of the sequences of E gene of 31 strains porcine Japanese encephalitis virus in GenBank was analyzed by DNAStar system. A pair of primers was designed according to the definite target sequences by Primer5 system.A 362bp specific strain was amplified by RT-PCR through the agar-gel electrophoresis. The digested RT-PCR products was two specific strains.Four Hebei isolates of JEV were detected by RT-PCR using the pair of primers. It was shown that all the four isolates could be amplified by RT-PCR. No amplification was observed when using RNAs extracted from HCV, PRRSV, and uninfected rat-brain or DNAs from PPV and PRV. However, 14 TCID₅₀ JEV could be detected by the technique. Thus, it was concluded that RT-PCR was more specific, sensitive and could be applied for diagnosis of JEV on molecular level.The method of SPA co-agglutination was conceived for diagnosis JEV from other viruses popularly, maneuverablely and inruitionisticly. We established the SPA plate co-agglutination test through making SPA combine hyperimmune serum. It was showed that this method was more sensitive, more specific and more reduplicate, furthermore it was handy, fast, not cost material and not requiring special apparatus, which was fit for fast identification of JEV in common field and epidemiological investigation.Reclaim and purification the protein antigen of JEV was searched. The molecular weight of the objective protein was determined by the SDS-PAGE aiming at the avian embryo virus, then reclaimed the objective protein by the horizontal electrophoresis. The titre of antiserum of cavain was ascertained by agar double diffusion and the positive result of antiserum was approved by the SPA co-agglutination. These results showed that the purified protein could induce well antibody response . The research developed the foundation for the single specific antibody.