Establishment And Early Warning Application Of Fluorescence Quantitative PCR Method For PEDV

Porcine epidemic diarrhea is a common contact intestinal infectious disease of pigs caused by porcine epidemic diarrhea virus,which has diarrhea and vomiting,dehydration and other feature in newborn piglets.Porcine epidemic diarrhea virus(PEDV)belongs to the Coronaviridae,Alphacoronavirus.At present,laboratory tests are the main detection methods,which mainly include immune-electron microscopy,immune-fluorescence,micro-serum neutralization test,enzyme-linked immune-sorbent assays,and routine RT-PCR techniques,but these methods have the defect like poor specificity,low sensitivity,and time-consuming operation,especially for the early diagnosis of the disease and the early warning of the epidemic.Fluorescent quantitative PCR has the advantages of high specificity,high sensitivity;good repeatability,and easy operation.The N gene of the PEDV genome is highly conserved,so this experiment selected the N gene as the target gene for the detection method.According to the complete sequence of the PEDV CV777 strain in GenBank,specific primers for the amplification of the full-length N gene were designed.In addition,a pair of specific primers was designed for fluorescence quantitative PCR to amplify a 190 bp fragment in length.The 750bp N gene was amplified from the positive samples using conventional RT-PCR.As a result of sequence alignment,the homology of the N gene sequence to the CV777 strain was 100%.Then the N gene was cloned into the pEASY-T1 simple cloning vector.After sequencing and identification,the correct recombinant plasmid was selected and diluted to 10-fold gradient as a positive standard and a fluorescence quantitative PCR standard curve,which was prepared to establish a rapid detection method of PEDV's content,which about SYBR Green I fluorescence quantitative PCR.The results showed a good linear relationship between the concentration of the reaction template and the Cq value.The established equation was Y=-3.287X+40.100,the correlation coefficient was 0.999,the amplification efficiency was 101.5%,and in the specificity test,the TGRV,PRRSV,CSFV and PRV were all negative in result.The sensitivity test showed that the detectable minimum concentration was 102 copies/?L,which was 100 times higher than the conventional RT-PCR method.The repeatability test showed that the coefficient of variation was 0.86%?1.68%.All the results indicated that the specificity,sensitivity and reproducibility of the SYBR Green I fluorescence quantitative PCR assay established in this experiment were all good.A total of 426 samples of anal swabs from multiple pig farms in Fuzhou,Putian,Quanzhou,and Sanming in Fujian Province were tested using the established detection method.The results showed that the positive rate of PEDV was 51.41%(219/426),indicating PEDV was common in pig farms in Fujian.Comparing PEDV positive rates in different farms,the highest positive rate in YS pig farms was 100%(104/104),and the lowest in QF farms was 17.78%(8/45);the positive rate of PEDV in different herds was compared.The highest positive rate of lactating sows was 68.00%(17/25),and the lowest in nursery herds was 46.03%(29/63),indicating that the difference in the positive rate of PEDV between different pig farms and different herds was greater.In this experiment,pigs were tested for PEDV in YLK,WY and YS three large-scale pig farms.Viral load data analysis showed that the viral load of samples recorded diarrhea exceeded 1.26×104 copies/?L.The diarrhea rate of suckling piglets in YS farms was severe,and the diarrhea rate was 40?50%.The viral load of the piglets in this farm was more than 1.26×104 copies/?L,which was 42.3%(11/26).The line was 1.26×103 copies/?L;the YLK pig farm had the highest viral load,exceeding 1.26×103 copies/?L,accounting for 63.63%,indicating that although there was no epidemic of diarrhea in this field,there was a great risk.At the same time,diarrhea piglets were necropsy in this study.PEDV was detected in different organs of diarrhea piglets.The results showed that the number of copies of PEDV in the intestine was significantly higher than other organs.Finally,in order to study the detoxification status of sows after PED immunization,the PEDV in the anal swab samples from 10 pregnant sows and 10 nursing sows at different time points after immunization was monitored.The results showed that on the 10th day,there were 3 pregnant sows's viral load exceeds 1.26×102 copies/?L,indicating that discharge of virus has begun;on the 15th day,viral load exceeds 1.26×102 copies/?L,3 of which exceed 1.26×103 copies/?L,indicating that the virus discharge peak has been reached in the period,the situation on the 30th day was the same as to that on the 15th day,indicating that the peak period of virus discharge was still present.On the 5th day,the nursing sows had two viral loads exceeding 1.26 × 102 copies/?L,indicating that virus discharge had begun;on the 15th day,all the viral load exceeded 1.26×102 copies/?L,of which one exceeds 1.26×103 copies/?L,indicating that the peak of virus discharge has been reached,on the 30th day,only one viral load exceeds 1.26×102 copies/?L,indicating that the peak period of virus discharge has passed.It was found that pregnant sows had a long virus discharge time after immunization.At this time,during the peak period of virus discharge,contact with piglets would cause exposure through contact and spread,which would make a reasonable PED for the farm.The vaccine immunization program provides a theoretical basis.In this study,quantitative real-time quantitative PCR was used to quantitatively detect PEDV in the collected samples.The data line with the clinical situation preliminarily established that the early warning line for the clinical symptoms of PED in suckling pigs was PEDV 1.26×103 copies/?L.The early warning of the epidemic is of great significance.In addition,the distribution characteristics of PEDV in various pigs and the dynamic trends of PEDV after sow immunization in PEDV were analyzed to understand the propagation direction and proliferation of PEDV.It was applied to farms to monitor the epidemic of PEDV,and provided a theoretical basis for further exploration of the PEDV early warning system and application.