Establishment Of Chemiluminescence Immunoassay For Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea(PED)is an acute and highly intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV).Since 2011,the mutated PEDV strain has spread throughout China,causing huge economic losses to China’s pig industry and also adding many challenges to the development of China’s modern pig industry.In clinical diagnosis,early and rapid detection of PEDV is particularly important to prevent the prevalence of PED.Chemiluminescence immunoassay(CLIA),as a new type of label detection technology,has extremely high sensitivity and a wide linear range.It is widely used in the field of diagnosis.At present,there is no report on the CLIA method for diagnosis of PED.In this study,the prokaryotic expression system was used to successfully express the first 150 amino acids and N protein of the structural protein PEDV,named N150 and N,respectively.These two proteins have been expressed in a soluble form,and the above two proteins were purified by affinity chromatography.Western-blot results showed that both purified proteins could react with positive sera infected with PEDV.N150 protein and N protein were used as coating antigens respectively,and a series of conditions of protein coating concentration coating conditions,blocking conditions,serum dilution,secondary antibody dilution and reaction time were optimized and successfully established two detection PEDV N The CLIA method of protein antibodies,namely N150-CLIA and N-CLIA.The detection of pig serum with known PED background showed that N150-CLIA(cut-off value 25434,diagnostic sensitivity 95.2%,diagnostic specificity 94.1%)was slightly higher than N-CLIA(cut-off value 22267,diagnostic sensitivity was 93.0%,specificity 94.6%).The inspection of 229 samples in the field showed that the coincidence rate between N150-CLIA and commercial imported ELISA kits was 91.7%,and the coincidence rate between N-CLIA and commercial imported ELISA kits was 89.1%.The stability of the two methods is analyzed,and the coefficient of variation between batches and within batches is less than 17%.In addition,the CLIA method has the advantage of rapid detection,which can be completed in 45 minutes.In this experiment,N150 and N protein were successfully expressed,and N150-CLIA and N-CLIA antibodies capable of rapidly detecting PEDV N protein were established.