Genetic Variation Of PEDV M Gene And Establishment Of Elisa Method For Detection Of PEDV Antibody

Porcine epidemic diarrhea(PED)is an acute intestinal disease caused by porcine epidemic diarrhea virus(PEDV),which is a major hazard to newborn piglets.It is widespread worldwide and caused colossal economic losses to the swine industry.At present,vaccination is the one of the most important measures for prevention and control the disease,but due to the variability of the virus in the long-term epidemic,the immune piglets still appear to be infected,and existing vaccines lose protection against new viruses.Therefore,it is of great practical significance to study the epidemic variation of the disease and develop a method for rapidly detecting antibody levels and assessing the immune effect.In this study,in order to analyze the genetic variation of epidemic diarrhea virus,M genes of PEDV epidemic strains isolated from Pig farms of Guangdong region were cloned and sequenced.Further,a competitive ELISA method for detecting PEDV antibodies was initially established.Research is as follows: 1?Genetic variation analysis of PEDV M gene:In order to analyze the genetic variation of epidemic diarrhea virus(PEDV)in Guangdong province from 2015 to 2018,M genes of 22 PEDV epidemic strains isolated from Guangdong region were cloned and sequenced.The results of sequence alignment and analysis showed high homologythat between the 22 PEDV isolates,but the homology among of the 22 PEDV isolates with the earlier domestic isolates CH/S and the classical strains CV777 was low.The results reveal that the current prevalent PEDV in Guangdong exists gene variation to form a unique evolutionary branch and has become the dominant strain.2?Establishment of PEDV antibody competition ELISA assay:The PEDV whole virus solution was used as the coating antigen,and the purified anti-PEDV egg yolk antibody was prepared in the laboratory as a competitive primary antibody.A competitive ELISA method for detecting porcine epidemic diarrhea virus antibody was established and the optimal reaction conditions were optimized to confirm the best.The threshold value for negative and positive result determination was calculated using the following formula:negative serum cut-off = X + 2SD = 32.5 %,positive serum cut-off = X + 3SD = 42.0%,between the two is suspicious.The coefficient of variation of intra-assay and inter-assay repeat was between 1.1% and 8.7%,both of which were less than 10%,which proved that the method was reproducible.The establishment of this method lays a good foundation for further development of ELISA antibody detection kit for PEDV.After further optimization of conditions,it is expected to be applied to large-scale PEDV epidemiological surveillance.