猪粪便中戊肝病毒实时荧光RT-PCR定性检测方法的建立及其分子流行病学特征

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

目的建立以TaqMan探针为基础的戊肝病毒(HEV)实时荧光RT-PCR检测方法和构建系统发育树,对戊肝病毒进行基因分型,检测中国部分省市猪场的猪粪便样本HEV污染水平。方法参照GenBank HEV基因型序列,针对HEV保守区设计引物和探针,优化反应体系,建立实时荧光RT-PCR和巢式RT-PCR检测体系。结果建立的HEV实时荧光RT-PCR检测方法的灵敏度为19.9拷贝/μL,扩增效率92.9%~109.1%,与札如病毒(Sa)、诺如病毒(Nov)、甲肝病毒(HAV)均不发生交叉反应。荧光RT-PCR检测猪粪便样本342份,其中HEV阳性样本210份,阳性率61.4%,育肥前阳性率56.6%,育肥后阳性率66.9%,育肥前后样本阳性率差异具有统计学意义(χ~2=24.8,P<0.05);经基因分型鉴定体系测定阳性毒株基因型均为HEV-4型,且存在4b、4d、4h三种基因亚型。结论中国部分省市猪场中HEV感染普遍,基因型均为HEV-4型,各省市猪场感染率和基因亚型存在差异。

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.METHODSDesigning 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.