[Establishment of a method with real-time reverse transcription-polymerase chain reaction for hepatitis E virus in swine feces and its molecular epidemiology].

OBJECTIVE:Real-time reverse transcription-polymerase chain reaction(real-time RT-PCR) assay based on Taqman and phylogenetic tree were developed for detecting hepatitis E virus in swine feces of pig farms from several provinces and city. METHODS:Designing prime and probe refering to HEV genotype sequences of Genbank, we developed a Taqman-based real-time RT-PCR assay and nested RT-PCR according to HEV conserved domain after optimizing reaction system, then detected the prevalence of HEV infection of pig farms. RESULTS:The sensitivity of the real-time RT-PCR assay established in this experiment was 19. 9 copies/μL, the amplification efficiency was 92. 9%-109. 1%, there was no cross reaction with Sapovirus, Norovirus and Hepatitis A. A total of 342 samples of swine feces were detected. There were two hundred and ten positive samples, and positive rate was 61. 4%. The positive rate of before-fattening was 56. 6%, and after-fattening was 66. 9%. The positive rate of before and after fattening samples had statistical difference(χ~2=24. 8, P<0. 05). The genotype identification system determined that the positive strains isolated in this study were HEV-4 type, and three subtypes of 4 b, 4 d and 4 h were detected. CONCLUSION:The pig farms of several provinces and city are contaminated by HEV extensively. The genotypes of the isolated strains are all HEV-4 type. The infection rate and infection subtype of pigs in different provinces and cities are different.