猪脑心肌炎病毒SYBR GreenⅠ real-time PCR检测方法的建立  

Development of a real-time PCR assay based on SYBR GreenⅠ for detection of porcine encephalomyocarditis virus

作　　者：施开创[1,2] 陈进喜[3] 屈素洁[2] 许瑞胜[3] 熊毅[2] 刘棋[2] 陈汉忠[3] 李刚[1] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100081 [2]广西动物疫病预防控制中心,广西南宁530001 [3]广西大学动物科学技术学院,广西南宁530005

出　　处：《中国兽医科学》2009年第2期135-139,共5页Chinese Veterinary Science

基　　金：广西科学基金项目(桂科青0728047);广西科技创新能力建设基金项目(08-05-01D)

摘　　要：针对猪脑心肌炎病毒(EMCV)VP1基因序列设计并合成了1对引物,以构建的含有该引物扩增序列的重组质粒作为阳性标准品,建立了检测EMCV核酸的SYBR GreenⅠreal-time PCR方法。检测结果显示,该方法线性关系好,标准曲线的相关系数达到0.991;对初始模板的检出下限为1×101copies/μL,比常规PCR方法高100倍;与其他猪源病毒,如口蹄疫病毒(FMDV)、猪生殖与呼吸综合征病毒(PRRSV)、猪瘟病毒(CSFV)、伪狂犬病病毒(PRV)均不发生交叉反应;组内与组间的变异系数均小于3%。应用建立的方法检测了17份临床可疑病例的心、脑组织样本,结果1份为阳性。表明,建立的real-time PCR检测方法灵敏度高、特异性强、重复性好,可以用于EMCV的病原检测及定量分析。A pair of primers was designed according to the sequence of the VP1 gene of porcine encephalomyocarditis virus（EMCV） and a recombinant plasmid containing the target gene was constructed as a standard control. Then,a real-time PCR assay based on SYBR Green Ⅰ for detection of EMCV was established. It had a good linear relationship between initial templates and Ct values,and the correlation coefficient of the standard curve was 0. 991. The assay had a detection limit of 1 × 10^1 copies/μL of initial templates,which was 100 times more sensitive than the conventional PCR. Moreover,the assay had no crossreaction with FMDV,PRRSV, CSFV and PRV, and had a coefficient of variations less than 3 % for both intra- and inter-assay. Seventeen heart and brain samples from suspicious cases in field were examined by the real-time PCR, and one was positive for EMCV. The high correlativity, sensitivity, specificity and reproducibility of this assay, together with its relatively rapid and simple procedure, indicated that the SYBR Green Ⅰ real-time PCR could be used as an effective assay for detection of nucleic acids and quantification of viral loads of EMCV.

关 键 词：脑心肌炎病毒 猪 荧光定量PCR SYBR Green Ⅰ 

分 类 号：S852.659.6[农业科学—基础兽医学]