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

作　　者：陈小金[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] 张俊哲[1] 

机构地区：[1]天津出入境检验检疫局动植物与食品检验中心,天津300467

出　　处：《中国动物检疫》2016年第7期72-77,共6页China Animal Health Inspection

摘　　要：对GenBank中基因3型猪戊型肝炎病毒（HEV）代表毒株基因进行序列分析,最终选定ORF3基因的部分序列为目的基因,运用生物信息学软件设计特异性引物和探针,扩增获得目的片段,并构建重组载体p UC-19-ORF3。梯度稀释重组质粒制备标准品,初步建立检测基因3型HEV的实时荧光定量PCR方法。对建立的检测方法进行特异性、敏感性和重复性验证。结果表明,标准品浓度在108～102 copies/μL范围内线性关系良好,相关系数为0.996。特异性试验结果显示,其它几种常见猪病病原体（猪流行性腹泻病毒、猪轮状病毒、猪传染性胃肠炎病毒、猪瘟病毒和猪繁殖与呼吸综合征病毒）未见典型扩增曲线。敏感性试验证实本方法的最低检测下限为101 copies/μL。重复性试验表明该方法对3个标准品检测结果的批内和批间变异系数低于2%。用该方法抽检59份进境猪肉样品,同时用普通RT-PCR方法对这些样品进行符合性试验,结果均为阴性。本研究所建立的检测方法不仅适用于进境猪肉样品中基因3型HEV的监控检测,而且还可用于基因3型HEV的早期临床诊断及分子流行病学调查。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 10^2 copies/μL to 108 copies/μ L, and the correlation coefficient of the assay was 0.996. The sensitivity for HEV was 10^1 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 HEMV.

关 键 词：猪戊型肝炎病毒 TAQMAN探针 荧光定量RT-PCR 

分 类 号：S851.3[农业科学—预防兽医学]