摘要
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.
作者
李延霞
江涛
李凤琴
彭子欣
王佳慧
李楠
张宏元
张立实
Li Yanxia;Jiang Tao;Li Fengqin;Peng Zixin;Wang Jiahui;Li Nan;Zhang Hongyuan;Zhang Lishi(West China School of Public Health,Sichuan University,Chengdu 610041,China;Key Laboratory of Food Safety Risk Assessment of Ministry of Health,China National Center for Food Safety Risk Assessment,Beijing 100021,China)
出处
《卫生研究》
CAS
CSCD
北大核心
2019年第4期545-551,559,共8页
Journal of Hygiene Research
基金
国家重点研发计划(No.2018YFC1602703)
关键词
戊型肝炎病毒
实时荧光RT-PCR
巢式RT-PCR
猪粪便
hepatitis E virus
real-time reverse transcription-polymerase chain reaction
nested RT-PCR
swine feces