Establish Of Real-Time RT-PCR Detection Assay For PEDV And Phylogenetic Analysis Of A Wild-Type Virus

Porcine epidemic diarrhea virus(PEDV),is one of the most common pathogens of porcine viral diarrhea outbreaks.After infecting piglets,it can cause porcine epidemic diarrhea(PED).The incidence and fatality rate of piglets during lactation is extremely high.Since 2010,PED has broken out in a large area in my country,so it is necessary for continuous monitoring of the prevalence of PEDV.In this study,the N gene of PEDV was connected to the p EASY-Blunt Simple vector to construct a standard plasmid.Using the T7 promoter on the plasmid,the RNA template which transcribed in vitro was a standard for fluorescent quantitative RT-PCR.The verification of sensitivity shown that the real-time fluorescent quantitative PCR can detect at least 10 copies.Through the detection of multiple viruses,the specificity and accuracy of the method are evaluated.The results shown that compared with other pathogens only PEDV can be amplified to the curve.Whether it is an intra-assay repeat test or an inter-assay repeat test,the coefficient of variation(CV)values of CT values were all below 2%,indicating that the method has strong specificity and high accuracy.161 samples collected in four regions of Shandong Province from 2018 to 2019 were detected by RT-PCR with the positive rate was 25.71-41.93%.This indicates that PEDV is very prevalent in the pig herds in the sampling area of this research,and its urgent for the prevention and control of the disease.Subsequently,this study carried out amplification sequencing and comparison analysis on the S sequence of PEDV epidemic strains form some areas of Shandong,and construct a genetic phylogenetic tree of the PEDV S gene.The result shown that those strains have a high related with the strains isolated in some areas of China recently,but the 6 strains are in two different groups,four G2 b type,one G2 a type and one G1 type.Except for SDLY1801,the other five strains have significant difference form the main vaccine strains,which indicating that the S gene sequence of the PEDV outbreak in the current breeding farm has been mutated during the epidemic.It is likely to cause the existing vaccine to lose its protective effect.In the end,Vero cells were used to isolate a PEDV epidemic strain SDLY1801 from the disease material,which transmitted on Vero cells for 5 generations.IFA testing confirmed that the PEDV strain was successfully isolated.According to the genome sequence of the reference strain,15 pairs of specific primers were designed,and the whole genome sequence of PEDV SDLY1801 strain was performed.Sequencing results shown that SDLY1801 belongs to the G1 type.There are no insertion or deletion mutations in the S gene,but there are many mutations,especially in the core epitope COE domain.Analyzing the antigenic index of the S gene,it was also found that there are differences in the antigenicity of the S protein of the SDLY1801 strain and the CV777 strain of amino acids between 20-100 aa and 300-350 aa.This shown that PEDV is constantly undergoing mutations and antigenic changes during the epidemic.This study also suggests that although PEDV infections in pig herds are dominated by G2 groups,the prevention and control of PEDV in G1 groups cannot be relaxed.