Epidemiological Investigation Of Porcine Epidemic Diarrhea And Establishment And Preliminary Application Of Rapid Diagnosis Methods

Porcine epidemic diarrhea(PED)is an acute,highly contagious intestinal infectious disease caused by porcine epidemic diarrhea virus(PEDV),which is characterized by vomiting,diarrhea,dehydration and high mortality of piglets.Pigs of different ages were all susceptible to PEDV infection.The small intestine was the main target for PEDV infection.Clinically,PEDV infection symptoms were similar with symptoms infected with gastroenteritis TGEV(Transmissible gastroenteritis virus comes)and PRo VA(porcine rotavirus A).It could not be identified under routine conditions,and must be confirmed by means of laboratory diagnosis method.Rapid and accurate diagnostic method is the important precondition for disease prevention and control.So,research on PEDV diagnosis method for pig epidemic diarrhea comprehensive prevention and control is of great significance.Since 2015 to 2017,to system was studied for the PEDV variation strains,we collected from nine pig 38 suspected diarrhoeal disease,and use the TGEV/PEDV/PRo VA triple real-time fluorescent RT-PCR detection kit for testing.For the results of the 15samples collected in 2015,all were PEDV positive,while the TGEV and PRo VA were negative.For the results of the 12 samples collected in 2016,8 were PEDV positive,4were PEDV and PRo VA positive.For the results of the 11 samples collected in 2017,7were PEDV positive,2 were PEDV,TGEV and PRo VA positive.Then,we select small intestine of only PEDV strong positive samples to try the separation of the virus in Vero-76cells.We implemented a PEDVstrain and named it as PEDV 2015-6.In order to achieve rapid,sensitive and specific detection of PEDV,we established a multiple real-time fluorescent RT-PCR detection method to differential diagnosis of PEDV,TGEV and PRo VA.According to the clinical application results,the coincidence rate of this method and commercialization kits reach 100%.This study developed a PEDV antigen fluorescence quantum dots immune chromatography test strip suitable for using in field detection.The coincidence rate of the test paper on the clinical application with the commercial was 94.74%(36/38).We developed a PEDV antigen fluorescence quantum dots immune chromatography test strip which is suitable for using in field detection.We use mouse monoclonal antibody against PEDV N protein 10A8a as quantum dots probes,and use monoclonal antibody10F2a as nitrocellulose membrane.By tested 50 negative controls,we determined the Cut-off fluorescence value of the test paper was 102 A.U..Further,we determined the best detection time of the test paper is 15 min.Sensitivity assay showed that the detection limit of the test paper for PEDV 2015-6 strain(10^7.5 TCID₅₀/ml)was 10^-6dilution(10^1.5TCID₅₀).Specificity assay showed that all test results are PEDV positive,while other common swine pathogen test results are negative.Repeatability study indicated that the relative standard deviations RSD of the test paper are less than 10%.Shelf life study indicated that the test paper can be placed at 37?for 28 d.The coincidence rate of the test paper on the clinical application with the commercial was 94.74%(36/38).In order to achieve the detection of Ig A antibodies in milk,we assembled the PEDV Ig A antibodies of quantum dots immune chromatography test paper.In order to evaluate on clinical application,we collected 120 pregnant sows'colostrum samples from 3 large-scale pig farms.Results show that 63 samples were positive.Furthermore,30 samples of positive and 30 samples of negative were randomly selected and detected by the test paper.The results showed that the positive coincidence rate of the two methods reached 93%.