Rapid Detection And Epidemiological Investigation Of Porcine Diarrhea Virus

Porcine diarrhea caused huge economic damage and seriously restricting the pig breeding industry development in China.Procine epidemic diarrhea virus,transmissible gastroenteritis virus and porcine rotavirus were the common etiologies for it in recent years.In this paper,a triple RT-PCR detection method established according to the uploaded sequence in GenBank.The method targeted at three conservative genes,such as PEDV N gene and TGEV N gene and PRoV VP7 gene.We optimized the triple RT-PCR detection method of the reaction system and conditions.The final results showed that the meathod we established had a good sensitivity and specificity.A colloidal gold rapid detection method for PEDV was established basing on the mAb-PEDV-2C11 and mAb-PEDV-1C12.We optimized the gold standard pads and sample pads,comparing the Nitrocellulose Filter Membrane species in the laboratory and choosing concentration of labeled antibodies.Finally we determined that the gold standard pad was treated with 0.02 M PB(pH=8.5),0.2%Tween-20,and 1.5%BSA solution.The sample pad was treated with 0.8%boric acid solution,and 1A/A NC film was selected as the final The NC membrane.The final concentration of tabeled antibody was 2 mg/mL.The detection showed that optimized detection was greatly improved about 7 times more sensitive than before.A method for the detection of PEDV by PLA(Proximity Ligation Assay)was preliminarily established.Two specificly probes were created by respectively binding the two existing monoclonal antibody mAb-PEDV-1C12 and mAb-PEDV-2C11 with Biotin B.The two probes could spontaneously combine to each other due to the two specificity antibody binding to the antigen.Those probes formed a template in the range less than 40nm.It could be detected by realtime-PCR,and the virus content in the disease can be determined with the detected negative and positive difference.Thus,the process of RNA extraction could be eliminated and the time of detecting the PEDV in the clinical samples was reduced.The final results showed that the method of PLA detection of porcine epidemic diarrhea virus could distinguish positive samples from negative controls.The method needed further optimized to meet the needs of clinical detecting.The number of all samples we collected from pig farms in Yangzhou,Yancheng,Lianyungang,Changzhou,Henan province,and Guangxi province was 982.All the samples were detected by using the established triple RT-PCR method.The result showed that the total PEDV positive detection rate was 9.1%(90/982),the TGEV positive detection rate was 0.1%(1/982),and the PRoV positive detection rate was 0.2%(2/982).Among them,the positive rate of a total of 80 samples from several farms in Yancheng,Jiangsu Province was 26.2%.It indicated that the current PED epidemic in China still seriously existing.At the same time,one case of PEDV and TGEV mixed infection was found in the detection of samples collected in Yancheng,and 2 cases of positive PRoV were found as well.They indicated that the conditions of mixed infections in the farms were not rare.We compared the detected PEDV ORF3 fragment with the NCBI uploaded sequences,and found that the major epidemic strains in Henan province were similar to the classic strains such as CV777 and DR13.The major epidemic PEDV nucleotide sequences in Jiangsu province were similar to the strains in USA,Thailand,and South Korea.Whcich showed that the prevalence of epidemic diarrhea had obviously differences at the present.