Establishment Of PCR-ELISA For Detection Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea is an acute,contagious intestinal infectious disease caused by porcine epidemic diarrhea virus.It can cause diarrhea,vomiting and dehydration in pigs,and the mortality rate of suckling piglets can reach 80%-100%.Since 2010,the outbreak of the disease in China has caused huge economic losses to pig farming industry.At present,the prevention and control of PEDV mainly includes comprehensive measures such as pathogen monitoring,immune control,biosafety,strengthening animal nutrition management,clinical diagnosis and so on.The PCR-ELISA method of pathogen is used for early monitoring of PEDV.The ORF3 gene sequence of PEDV published by GenBank was compared and analyzed.A pair of RT-PCR specific primers were designed and synthesized for conservative sequence.The target gene fragment of PEDV ORF3 was amplified by PCR and cloned into pMD19-T vector.The recombinant plasmid of pMD19-T-ORF3 was constructed.The target gene was identified by PCR and sequencing.Digoxin and biotin were labeled at the 5'end of the upstream and downstream primers,and a pair of primers with markers were synthesized.The constructed plasmid was used as template,and the primers with markers were used for PCR amplification under the same conditions,and the products with markers at both ends were obtained.According to the operation steps of PCR-ELISA,the encapsulation concentration of streptavidin used in PCR-ELISA,the blocking concentration of BSA solution,the reaction time of PCR products,the working dilution concentration of enzyme-labeled antibody,the reaction time and the colour developing time were optimized,and then the optimum conditions were determined.Thirty-five samples with known negative PEDV were detected by PCR-ELIAS.The OD450 nm value of the samples greater than or equal to 0.35 was positive and less than 0.29 was negative,which was suspicious between the two samples.The genomic cDNA of PEDV was diluted and detected by PCR-ELISA to determine its sensitivity.PRRSV,PRV,CSFV,PCV-2 and Japanese B encephalitis DNA were used as templates to detect the specificity of PCR-ELISA under the best reaction conditions.The stability of the method was determined by detecting the intra-batch repeatability and inter-batch repeatability of the enzyme labels coated at the same time with the same disease material and at different times with the enzyme labels coated at the same time.The results were compared with those of RT-PCR and real-time fluorescence quantitative PCR.The results showed that the band size of conventional PCR was 227 bp.Sequencing analysis showed that the band had 100%homology with PEDV ORF3 gene published on GenBank,which proved that the target fragment was PEDV ORF3 gene.The optimum reaction conditions of PCR-ELISA were as follows:1:800 diluted streptomycin?1mg/mL?was coated overnight at 4?,2%BSA was sealed at 37?for 15 min,1:50 diluted PCR product was treated at 37?for 15 min,2 000 times diluted enzyme-labeled antibody reacted at 37?for 15 min,and colour reagent developed for 5 min.The critical value of 35 samples negative for PEDV detected by PCR-ELIAS was 0.35,and the OD_450nm value of 35 samples was positive or negative.The lowest detection limit of PCR-ELISA 3.94×10³copies/?L can be detected in the positive plasmids established by this experiment.The lowest limit detected by conventional PCR is 3.94×10⁶copies/?L.The results show that PCR-ELISA sensitivity is1000 times higher than that of conventional PCR,and it has no cross reaction with PRRSV,PRV,CSFV,PCV-2 and Japanese B encephalitis,indicating its specificity.Of 64 clinical samples,31 were positive by PCR,the positive rate was 48.4%.36 were positive by PCR-ELISA,the positive rate was 56.2%.36 were positive by fluorescence quantitative PCR.Five samples identified as negative by PCR-ELISA and positive by fluorescence quantitative PCR were identified by virus isolation,culture and IFA.The results showed that the established PCR-ELISA method had high accuracy,and the coincidence rate with fluorescence quantitative PCR was 100%.In conclusion,an efficient,rapid,specific,sensitive and safe PEDV detection method has been established in this experiment,which provides technical support for further assembly of kits or development of test strips for epidemiological investigation,environmental monitoring and diagnosis of PEDV in pig farms.