猪脑心肌炎病毒TaqMan荧光定量RT-PCR检测方法的建立及应用  

Development and application of a TaqMan real-time RT-PCR assay for the detection of porcine encephalomyocarditis virus

作　　者：施开创[1] 陈宏备[2] 覃芳芸[1] 李向涛[2] 胡杰[1] 郑喜邦[2] 李军[1] 

机构地区：[1]广西动物疫病预防控制中心,广西南宁530001 [2]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》2011年第9期928-932,共5页Chinese Veterinary Science

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

摘　　要：为了建立猪脑心肌炎病毒(EMCV)的检测方法,根据GenBank中EMCV 3D基因的保守序列设计并合成1对引物和1条TaqMan探针,经过反应条件的优化,建立了该病毒的TaqMan荧光定量RT-PCR检测方法。该方法线性关系好,以重组质粒标准品构建的标准曲线的相关系数达到0.998;敏感性高,检测下限为10copies/μL,比普通PCR敏感100倍;特异性强,只有EMCV呈阳性,而与猪口蹄疫病毒、猪瘟病毒、猪生殖与呼吸综合征病毒、猪伪狂犬病病毒、猪细小病毒、BHK-21正常细胞均无交叉反应;重复性好,组内与组间的变异系数均小于1.5%。应用所建立的检测方法,对猪源EMCV GXLC株人工感染小鼠的脑、心、脾中的病毒载量进行了检测,同时对临床采集的153份可疑病料进行了检测。结果表明,本研究建立的TaqMan荧光定量RT-PCR检测方法灵敏度高、特异性强、重复性好,可以用于EMCV的检测及定量分析。A TaqMan real-time RT-PCR assay was established for the detection of porcine encephalomyocarditis virus（EMCV）.The assay utilized a pair of specific primers and a TaqMan probe which was based on the conserved region of the 3D gene of EMCV.The established assay possessed a fine linear relationship between initial templates and Ct values,and the correlation coefficient of the standard curve was 0.998.The detection limit of the method was 10 copies/μL of initial templates,which was 100-fold more sensitive than that of the conventional PCR.And the method was also highly specific with the fluorescent signals only be detected from EMCV,but not from foot-and-mouth disease virus,classical swine fever virus,porcine reproductive and respiratory syndrome virus,porcine pseudorabies virus,porcine parvovirus and normal BHK-21 cells.Moreover,the method was highly reproducible and had a coefficient of variation less than 1.5% for both intra-and inter-assays.The established assay was successfully applied for the detection of viral loads in the brain,heart and spleen tissues from mice which were experimentally infected with porcine EMCV GXLC strain,and for the detection of viral nucleic acids in 153 clinical samples collec-ted from suspected EMCV-infection piglets.The high sensitivity,specificity and reproducibility of the assay indicated that the TaqMan real-time RT-PCR assay could be used as an effective tool for the detection and quantification of EMCV.

关 键 词：脑心肌炎病毒 猪 荧光定量反转录聚合酶链反应 TAQMAN探针 

分 类 号：S852.659.6[农业科学—基础兽医学]