Establishment Of RT-PCR Detection Method For Porcine Japanese Encephalitis Virus And JEV Isolated From Sichuan PrM/E Gene Sequences Analysis

Japanese encephalitis is one kind of central nervous system's acute infectious diseases after mosquito bite seriously threaten to human and animal health. The pig infected JEV is Japanese encephalitis's important host and amplification of the host, and is JEV's main source of infection. This disease caused a severe hazard to graziery. In this study, the rapid diagnosis of JE method was established and new isolates of JEV from ChongZhou and NeiJiang pig farms in Sichuan province,which were analyzed according to PrM/E gene nucleotide sequences. The study know the molecular characteristics of two new isolates of JEV from molecular level, this might be of great significance for epidemiology and control researches of Japanese encephalitis.1. Establishment of RT-PCR detection method for porcine Japanese encephalitisIncluded reference to GenBank, the 31 JEV E gene, using homologous DNAMAN software analysis to SA14-14-2 1725～2163 for the reference section to determine the amplification of target sequences; synthesized using Primer 5 software, a pair of primers (P1/P2), product length is 439bp.To optimize the reaction conditions by choosing Mg2+ concentration,primer concentration and annealing temperature.From the results of optimization,JEV PT-PCR amplification of the best system:10×buffer (Mg2+-Free) 2.5μl, 25mM Mg2+ 1μl,2.5mM dNTP 2μl, 10pmol of upstream and downstream primer 0.8μl, 5U/μl Taq DNA polymerase 0.5μl, DNA template 2μl, the last with sterile deionized water up to 25μl. PCR reaction program predegeneration 94℃for 3 min, then 94℃denaturation 1min,55-57℃renaturation between the 45s,72℃extension of 1min, a total of 30 cycles, final extension of 72℃in 10min,4℃preservation. JEV RT-PCR for specific detection:the common classical swine fever virus, porcine reproductive and respiratory syndrome virus, porcine pseudorabies virus,porcine parvovirus virus was amplified, no band; It could detected the volume of 10pg of DNA for susceptibility testing.Experiments show that:In this study, the RT-PCR method of detection of JEV was established, the detection method have the advantages of simple, quick, good specificity and high sensitivity detection of JEV.2. JEV CZ1 and NJ1 isolated from Sichuan PrM/E gene sequence analysisThe PrM/E gene nucleotide sequences amplified and cloned from the new isolated strains,the genotype analyzed according to PrM(456-695) sequences with the referenced sequences of 29 strains,and the E nucleotide sequences compared with the SA14-14-2 live attenuated vaccine strain.The results show that:The JEV-CZ1 was grouped into genotypeⅠof JEV,the homology of the nucleotide and amino acid sequence between the isolated strain and vaccine stain was 90.9% and 97.2% respectively,but there were amino acid substitutions in 15 sites of the key active domains; The JEV-NJ1 was grouped into genotypeⅢof JEV,the homology of the nucleotide and amino acid sequence between the isolated strain and vaccine stain was 99.6% and 99.2% respectively,but there were amino acid substitutions in 3 sites of the key active domains.The molecular characterization was preliminarily confirmed.This might be of great significance for epidemiology and control researches of Japanese encephalitis.