猪血凝性脑脊髓炎病毒SYBR Green Ⅰ实时荧光定量PCR检测方法的建立  

Development of a SYBR Green Ⅰ real-time PCR assay for detection of porcine hemagglutinating encephalomyelitis virus

作　　者：臧德跃[1] 王栋[1] 李明谦[1] 赵魁[1] 张冰冰[1] 陆慧君[2] 贺文琦[1] 陈克研[1] 岳占碰[1] 高丰[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062 [2]吉林大学人兽共患病教育部重点实验室人兽共患病研究所,吉林长春130062

出　　处：《中国兽医科学》2011年第1期60-64,共5页Chinese Veterinary Science

基　　金：国家自然科学基金资助项目(30871849;31072134);吉林省科技发展国际合作项目(20080722);吉林省青年科研基金项目(20090154)

摘　　要：根据GenBank中的猪血凝性脑脊髓炎病毒(HEV)S蛋白基因的保守序列设计合成1对特异性引物,经PCR扩增S基因并构建含有该基因片段的重组质粒,将该重组质粒作为阳性标准品,建立了检测HEV的SYBR Green Ⅰ real-time PCR方法。结果显示,该方法线性关系良好,相关系数为0.994;敏感性高,检测下限为1.6×104 copies/μL;特异性强,与牛冠状病毒、伪狂犬病病毒和猪繁殖与呼吸综合征病毒不发生交叉反应;并且重复性好,组内、组间的变异系数均小于2%。应用该方法对采集的20份疑似HEV感染病料进行检测,其中16份为阳性,而用常规PCR检测同样的样品,仅8份为阳性。表明建立的SYBRGreen Ⅰ real-time PCR方法具有快速、特异、敏感、重复性好等优点,可用于临床上HEV的检测及定量分析。With a pair of primers designed according to the conserved sequence of S gene of hemagglutinating encephalomyelitis virus（HEV） in GenBank,the S gene was amplified by PCR and the recombinant plasmid containing the gene was constructed as a standard control.Then,a real-time PCR assay based on SYBR Green Ⅰ for HEV detection was developed.In result,the developed assay had a good linear relationship between initial templates and Ct values,and the correlation coefficient of the standard curve was 0.994.The assay had high sensitivity and could detect 1.6×104 copies/μL.The specificity of the assay was high,so there was no cross reaction between HEV and other pathogens including bovine coronavirus,pseudorabies virus,porcine reproductive and respiratory syndrome virus.In addition,the assay had good reproducibility and there was a coefficient of variations less than 2% for both intra-and inter-assay.16 out of 20 suspicious positive samples detected by the real-time PCR were positive,while 8 out of the 20 suspicious positive samples detected by the conventional PCR were positive.These results showed that the developed SYBR Green Ⅰ real-time PCR assay had the advantages of rapid detection,specificity,sensitivity and good reproducibility,and it could be applied for the clinical diagnosis and quantification analysis of HEV.

关 键 词：猪血凝性脑脊髓炎病毒 S蛋白基因 荧光定量聚合酶链反应 

分 类 号：S852.659.6[农业科学—基础兽医学]