Establishment Of PEDV-TGEV-PRoV Triple Detection Protein Chip

Porcine epidemic diarrhea virus(PEDV),porcine transmissible gastroenteritis virus(TGEV),porcine rotavirus(PRoV)are the three major viral pathogens of diarrhea in pigs.These pathogens are susceptible to pigs of all ages,especially can cause very high mortality rate of young piglets.The mixed infection of these three viruses occurs frequently,and leads to much more serious damage of the intestinal tract than the single infection.The clinical symptoms and pathological changes of the three viruses are very similar,which brings many difficulties to the diagnosis and prevention of the disease,causing huge economic losses to China and other pig-raising countries in the world.Efficient and fast methods for identifying these three viral pathogens can quickly confirme the cause of diarrhea in pigs,which contributes to the treatment of this diease,and the reduction of damages caused by the disease.At present,the methods for detecting these three viruses mainly include RT-PCR method,ELISA method and colloidal gold test strip method.The colloidal gold test strip method is fast,but the sensitivity is low;the ELISA method has good specificity,high sensitivity and simple operation,but only one pathogen can be detected at a time;although the RT-PCR method can identify the three pathogens,the operation is cumbersome,time-consuming,labor-intensive,professional,and easy to be polluted.Therefore,the establishment of a specific,sensitive,rapid and efficient detection method that can simultaneously detect and distinguish these three pathogens is of great significance for clinical diagnosis.This study intends to establish a highly specific,sensitive,and high-throughput detection method,based on the principle of double antibody sandwich detection antigen.This method is a kind of protein chip,using PEDV,TGEV,PRoV monoclonal antibodis as captural antibodis and labeled antibodis.This protein chip method will provides a new technical mean for the differential diagnosis of porcine viral diarrhea caused by PEDV,TGEV and PRoV.For the first step,the hybridoma cell lines of PEDV,TGEV,and PRoV monoclonal antibodis preserved in the laboratory were resuscitated,then the monoclonal antibodis were purified and labeled by HRP after ascites preparation,and subsequently the purity and concentration of the prepared monoclonal antibodis were determined.Based on the double antibody sandwich principle(monoclonal antibody-antigen-enzyme labeled monoclonal antibody),the PEDV-TGEV-PRoV triple detection protein chip was constructed by using the prepared monoclonal antibodis with denatured silicone membrane as the carrier.And the detection conditions of this protein chip like the pairing mode of the three monoclonal antibodies,the reaction mode,and the optimal concentration of the captural antibodis,the optimal dilution of the enzyme-labeled antibodis,the reaction time,and the color development time were optimized.The specificity,sensitivity,repeatability and methodological control of the protein chip were evaluated based on the determination of the detection conditions.The captural antibodies used in this study were identified as monoclonal antibodies PEDV-8A3 strain,TGEV-4B4 strain,and PRoV-3A8 strain.The enzyme-labeled antibodis were monoclonal antibodis PEDV-12C4 strain,TGEV-5H11 strain,and PRoV-3A8 strain.The optimal concentrations of captural antibodies were PEDV-8A3 0.8 mg/mL,TGEV-4B4 0.6 mg/mL,and PRoV-3A8 0.6 mg/mL.The optimal dilution of the enzyme-labeled antibodis were 1:1500 for PEDV-12C4,1:1500 for TGEV-5H11,and 1:3000 for PRoV-3A8.The reaction mode was one-step method,the reaction time was 45 min,and the color development time was 15 min.Then 100 negative clinical samples were tested to determine the decision threshold,and the results suggested that PEDV was 3392,TGEV was 3147,and PRoV was 3936.The PRV,PPV,PCV2,CSFV and PRRSV positive samples were tested to evaluate the specificity of this protein chip,and the results showed no cross-reaction.The sensitivity of this protein chip to the three viruses was tested by detecting these three viruses with diffirent dilutions,and the results showed as 80 times dilution of PEDV 107.0TCID50/mL virus solution,160 times dilution of TGEV 107.5TCID50/mL virus solution,and 20 times dilution of PRoV 106.5TCID50/mL virus solution.The intra-and inter-assay repeatability of the protein chip was within 15%,suggesting the repeatability was good.This study established a rapid detection method of PEDV,TGEV and PRoV based on protein chip technology.Its further improvement and popularization application have important value for rapid diagnosis,effective treatment and prevention of diarrhea caused by PEDV,TGEV,and PRoV.