Molecular Epidemiological Investigation Of PEDV Variants And Establishment Of Dual RT-PCR Differential Detection Methods For Three Kinds Of Virus Strain

Porcine epidemic diarrhea virus(PEDV),is one of the most common pathogenic pathogens that cause diarrhea clinically in piglets.Since the disease was discovered in 1971,it has been endangering the entire pig industry and causing severe economic losses to our country.In this study,in order to grasp the genetic variation of the S gene of PEDV epidemic strains in multiple regions of Shandong,and to establish a differential diagnosis method for PEDV vaccine strains,mutant strains and classic strains,the established differential diagnosis method was used to detection and identification clinically large quantities of samples.The studies design 4 pairs of specificity for the PEDV S gene by duplex RT-PCR method.The primers were used to amplify the S gene,analyze its homology,construct a genetic phylogenetic tree of the PEDV S gene,and design two pairs of primers for different regions of the ORF3 gene deletion site and the S gene mutation region to complete the differential diagnosis of the three PEDV strains.The test results show that the overall positive rate of suspected PEDV disease materials is 76.40%.The homology analysis of the 8 PEDV mutants obtained in this study indicated that there were insertions,deletions,and mutations in the S gene sequence of the 8 mutants.And in the genetic evolution tree are located on three large branches,there are large differences between the various strains.The duplex RT-PCR method established in this research successfully optimized the reaction conditions to successfully amplify the expected gene fragment of 149 bp,198 bp,and 429 bp.The identification of three PEDV strains can be completed in the same RT-PCR reaction system.The repeatability test proves that the method is stable,cross-reactivity detection has no cross-reaction phenomenon,random test results of clinical samples and single RT-PCR test results are consistent.This study reached the following conclusions: PED still has great harm to the swine industry in Shandong.Through comparison,it is found that the S gene of PEDV epidemic strains in Shandong has diversity in genetic variation and different homology.Using the PEDV duplex RT-PCR method established in this study to detect three strains accurately against vaccine strains,mutant strains,and classic strains Judging that the mutant strain accounted for 77.54% of PEDV-positive strains,it has become the dominant strain in pig herds.In the daily breeding and prevention and control work,the harm of PEDV to pigs should be minimized,and the necessary epidemic prevention work should be strengthened while the detection and molecular epidemiological investigation of the three PEDV strains should be strengthened in a timely manner.The development trend of the epidemic,formulate scientific preventive measures to ensure the sustainable and healthy development of the herd.