Molecular Epidemiology And Development Of Indirect Elisa Method Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea (PED) is an acute and highly contagious disease in swine. The pathogenic agent of this disease was identified for the first time. Since the end of2010,the intensity and epidemic area of PED have continuously enlarged, and it causes a high mortality of suckling piglets and serious economic losses to our country’s pig industry.At present, There is no standardized diagnostic methods which can be Widely used At the grassroots level in the world. therefore,further epidemiological investigation of domestic pandemic strain and the decelopment of fast and effective diagnostic methods to PEDV is necessary and significant for PED prevention and control. In this study, RT-PCR detection method aimed at M gene and indirect ELISA based on recombinant protein S582were respectively developed and used for epidemiological survey of PEDV. It has practical significance for the further research of diagnosis and pathogenesis of the disease. The main contents were as following:1. Dvelopment and application of RT-PCR for detecting PEDVAn RT-PCR method for PEDV conservative gene M was developed and confirmed to be very specific and sensitive. During the period of2011-2012,111diarrhea samples were collected from25pig farms and PEDV was detected by the RT-PCR method. The results showed that70samples from20pig farms were positive. The farm positive rate was83.33%and the sample positive rate was61.95%. It suggested that, PED should be paid more attention because PEDV is still one of the main pathogens of the diarrhea.2. Prokaryotic expression of PEDV partial S proteinAccording to the sequence in GenBank, partial S protein gene of PEDV was amplified and cloned into pET-28a to construct a recombinant plasmid named pET-28a-S582. After identifying and transforming of pET-28a-S582, an27KD fusion protein was expressed in BL21. Western-blot tests showed high stability and reactogenicity with anti-PEDV serum of the ptotein.The result laid the foundation for development of diagnostic kits and genetic engineering vaccine.3. Dvelopment and application of indirect ELISA based on recombinant protein S582An indirect ELISA method was established with the purified recombinant protein S582as the coated antigen through optimization in reaction conditions. The optimal antigen concentration was set at4μg/mL. The coated time was2h at37℃. The sealing buffer was1.5%BSA-PBST. The best dilution of serum and HRP-SPA were1:200and1:10000,and the reaction time was lh and30mins at37℃respectively. The Incubation time of TMB substrate was defined as10mins at37℃. The cut-off value of ELISA was determined by detecting30PEDV negative sera. And the diagnostic accuracy was94.79%compared with TSZ ELISA kit on96serum samples.Cross-reactivity assay showed that S582ELISA was PEDV-specific. It indicated that the S582ELISA is not only sensitive, specific, and repeatable but also suitable for large scale epidemiological surveys of PEDV infection.