Establishment Of Fluorescent Quantitative PCR Assay For Swine Hepatitis E Virus And Molecular Epidemiology In Yancheng Region

Hepatitis E caused by Hepatitis E virus(HEV) is an acute viral infectious disease. HEV is mainly prevalent in developing countries, sporadic prevalent in industrialized countries. It has a higher infection rate in our country and very few drugs for the treatment of hepatitis on the market. Now in order to prevent and control HEV, mainly take measures to prevent and cut off the the route of transmission.1. To establish a rapid and sensitive detection method of swine hepatitis E virus(SHEV), the Taq Man real-time PCR was developed with a pair of primers and the probe targeting the conserved region of HEV ORF2. With the optimized reaction conditions, the standard curve showed a linear relationship from 102 to 109 copies/μL with the correlation coefficient(R2) of 0.999. Specificity tests showed the assay had no cross reaction with porcine circovirus type 2, classical swine fever virus, porcine reproductive respiratory syndrome virus and pseudorabies virus. Sensitivity tests showed that the detection limit was 101 copies/μL. Repeatability tests showed that the the coefficient of variations of threshold cycles were than 3.0% for both intraand intro-assay. The 131 clinical samples collected from Hei Longjiang province(45 liver samples and 86 feces samples of pigs) were detected by the assay, and the total positive rate was 11.45%(22.22% from liver samples and 5.81% from feces samples). The established real-time PCR assay had a potential application for rapid and quantitative detections of SHEV.2. In order to research the prevalence of SHEV infections among Yancheng pig farms, 747 pig fecal samples were collected from 10 pig farms located in Yancheng of Jiangsu province. Adopt the method of RT-nPCR to amplification the target fragment of SHEV RNA. All of ten pig farms dected positive samples.The prevalence of HEV infection within the different districts and growth stage varied between 2% and 28%.The research results show that 86 samples to be HEV positive, 81 samples can be sequenced. Compare with the results of sequencing analysis, build the system evolutionary tree, homologous evolution analysis. Phylogenetic analysis indicated that 73 isolates belonged to HEV genotype 4. The remaining 8 isolates belonged to genotype 3.