Establishment Of One-Step Fluorescent Quantitative PCR Detection Assay And Genetic Evolution Analysis Of Porcine Epidemic Diarrhea Virus

Porcine epidemic diarrhea virus(PEDV)belongs to the coronavirus family,which can cause severe diarrhea and high mortality in newborn suckling piglets.Since 2010,the severe outbreak of PEDV variant has caused huge economic losses to the pig industry.Therefore,it is necessary to quickly and comprehensively understand the genomic characteristics and cell infection characteristics of porcine epidemic diarrhea virus,so as to provide reference for the diagnosis and prevention and control of porcine epidemic diarrhea,ensure the control of the disease in a short time,and avoid causing serious economic losses.In this study,a real-time fluorescence quantitative PCR detection method for PEDV was established,and this method was used to detect piglet diarrhea samples collected from farms in Henan Province.Finally,7 strains of PEDV were successfully isolated,and 4 complete genome sequences were obtained by metagenomic sequencing,and their genetic evolution and amino acid mutation sites were analyzed.1.Establishment of one-step fluorescence quantitative PCR detection method for PEDVIn order to establish a rapid diagnostic method for PEDV,a pair of specific primers were designed according to the conserved region of PEDV Rd Rp gene,and a real-time fluorescence quantitative PCR detection method for PEDV was established.The results showed that the minimum detection limit of the method established in this experiment was6.8 copies / μL,while the minimum detection limit of ordinary PCR was 6.8 × 101 copies /μL,and the sensitivity was 10 times higher than that of ordinary PCR.In addition,no fluorescence signal was detected in the detection of PDCo V,TGEV,SADS-Co V and other pathogens,and the coefficient of variation within and between groups was less than 2 %.This study successfully established a SYBR Green I real-time fluorescence quantitative PCR detection method for PEDV,which has good specificity and repeatability.It provides technical support for rapid diagnosis,prevention and molecular epidemiological detection of PEDV.In addition,on this basis,this study also tried to use a nucleic acid lysate to obtain viral RNA directly from the disease material,which only took 20 minutes,simplifying the steps and shortening the time,avoiding the errors generated during the sampling process.Compared with the conventional method of obtaining viral RNA,the results showed that both methods could detect PEDV.2.Isolation and identification of porcine epidemic diarrhea virus and genetic evolution analysis of S geneThe PEDV-positive piglet diarrhea material was treated and inoculated into Vero cells,and 7 PEDV strains of HNZK1,HNZK2,HNZK3,HNZK5,HNLY,HNXH and HNYZ were successfully isolated.The S gene sequencing,S gene sequence homology comparison and genetic evolution analysis of 7 PEDV strains were carried out.The results showed that the nucleotide homology and amino acid homology between the seven strains were 96.9 %-99.9 % and 96.4 %-99.7 %,respectively.The homology of nucleotide and amino acid was97.0 %-98.4 % and 97.0 %-98.5 %,respectively.The homology of nucleotide and amino acid was 95.0 %-96.4 % and 94.2 %-96.2 %,respectively.The results of genetic evolution analysis showed that 7 PEDV strains belonged to GII genotype.Subsequently,multiple sequence alignment analysis of the amino acid sites of the S gene revealed that eight additional mutations occurred in the S protein of HNZK1 and HNZK2 strains.The mutation sites were N29 K,Y52F,P155 S,N383Y,Q827 H,S889R,V892 M,V1085I,and the first three were located in the S1-NTD region;in addition,the GIIb strain had specific mutations in T369 I,R492T,I501 T,V848A,F1212 Y,S1220D and P1270 S.The GIIa strain has specific mutations in G1364 C and A1381 V.This study will provide a theoretical basis for the genomic characteristics and evolutionary trends of the latest PEDV strains in Henan.3.Genome sequencing and analysis of porcine epidemic diarrhea virus Henan isolatesIn order to understand the variation characteristics of PEDV isolates,the whole genomes of HNZK1,HNZK5,HNXH and HNYZ isolates were sequenced by meta-genomic sequencing technology.The results of sequence analysis showed that the full-length gene sequence of HNZK1 was 28033 bp,the full-length genome of HNZK5 was 28013 bp,the full-length genome of HNXH was 28038 bp,and the full-length genome of HNYZ was28038 bp.The analysis of the main structural genes showed that the M,N and E gene sequences of the isolates were consistent,and the lengths of S and ORF3 genes were different.The full-length S gene of HNZK1 strain was 4158 bp,and the other three strains were 4161 bp;the ORF3 gene of HNZK5 strain was 654 bp,and the other three strains were675 bp.Further amino acid sequence alignment analysis of the structural proteins of the isolates with reference strains at home and abroad revealed that there were a large number of deletions in ORF3,and M,E,and N proteins were relatively conserved.Recombination analysis of the whole genome sequences of HNZK1,HNZK5,HNXH,and HNYZ revealed that recombination events may occur between strains of GIIa and GIIb genotypes.These conclusions will help us understand the inheritance and variation of PEDV and provide a theoretical basis for PEDV genetic evolution.