中国动物检疫2016，Vol.33Issue(7)：72-77,6.DOI:10.3969/j.issn.1005-944X.2016.07.023
基因3型猪戊型肝炎病毒荧光定量RT-PCR检测方法的建立
Establishment of TaqMan Fluorescence Quantitative RT-PCR Assay for Swine Hepatitis E Virus Detection
陈小金 1张俊哲 1董志珍 1赵祥平 1王建华 1刘洋 1王玉玲 1陈本龙 1肖妍 1王乃福1
作者信息
- 1. 天津出入境检验检疫局动植物与食品检验中心，天津 300467
- 折叠
摘要
Abstract
Primers and probe were designed based on the ORF3 gene sequences of genotype 3 Hepatitis E virus (HEV)available in GenBank. A recombinant plasmid pUC19-ORF3 containing the target gene was constructed as a standard control,then a real-time PCR assay for the detection of HEV was established. The specificity,sensitivity and reproducibility of the assay were evaluated and compared with conventional RT-PCR. Results showed that the real-time RT-PCR assay was highly specific with a broad linear detection range from 102 copies/μL to 108 copies/μL,and the correlation coefficient of the assay was 0.996. The sensitivity for HEV was 101 copies/μL. The specificity analyses using cDNA of porcine reproductive and respiratory syndrome virus,classical swine fever virus,transmissible gastroenteritis virus of swine,porcine rotovirus and porcine epidemic diarrhea virus as the templates that the amplifications from these viruses were negative while the amplification from HEV were positive. Inter-and intra-assay variables of CV were less than 2%. 59 samples detected by the real-time PCR and RT-PCR were negtive with HEV. The coincidence of the real-time PCR was 100%with RT-PCR for the samples. The results showed that the method established in this study was suitable for detecting the genotype 3 HEV of imported pork samples. This assay also provided an effective method for rapidly diagnosis,vaccine quality control and molecular epidemiological investigation of HEV.关键词
猪戊型肝炎病毒/TaqMan探针/荧光定量RT-PCRKey words
Hepatitis E virus of swine/TaqMan probe/fluorescence quantitative RT-PCR分类
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陈小金,张俊哲,董志珍,赵祥平,王建华,刘洋,王玉玲,陈本龙,肖妍,王乃福..基因3型猪戊型肝炎病毒荧光定量RT-PCR检测方法的建立[J].中国动物检疫,2016,33(7):72-77,6.
