小鼠IL-6、iNOS TaqMan荧光定量PCR检测方法的建立及应用  

Development and Application of a TaqMan Real-time PCR Assay for Detection of Mouse IL-6 and iNOS

作　　者：施开创[1] 陈宏备[2] 胡杰[1] 覃芳芸[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：《中国畜牧兽医》2011年第9期44-51,共8页China Animal Husbandry & Veterinary Medicine

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：为探讨脑心肌炎病毒(EMCV)感染后IL-6、iNOS基因的表达水平,从分子水平深入研究EMCV的致病机制,分别针对小鼠IL-6、iNOS及管家基因β-actin的基因序列设计特异性引物和探针,构建含有各自引物扩增序列的重组质粒作为阳性标准品,建立了检测IL-6、iNOS和β-actin的TaqMan rea-ltime PCR检测方法。该方法线性关系好,IL-6、iNOS和β-actin标准曲线的相关系数均达到0.998;敏感性高,初始模板的检出下限均达到1×101拷贝/μL;特异性强,只有以总RNA反转录成的cDNA为模板,并加入特异性引物和探针的反应才检测到荧光信号;重复性好,组内与组间的变异系数均小于2%。应用所建立的检测方法,对猪源EMCV GXLC株感染小鼠的脑、心脏中IL-6、iNOS mRNA的表达水平进行了检测。结果表明,本研究建立的TaqMan real-time PCR检测方法灵敏度高、特异性强、重复性好,可以用于小鼠IL-6、iNOS基因的检测及定量分析。A pair of primers was designed according to the sequence of mouse IL-6 and iNOS genes as well as housekeeping gene β-actin,respectively,and recombinant plasmids containing the target gene was constructed as a standard control for IL-6,iNOS and β-actin.Then,the real-time PCR assays based on TaqMan probes for detection of IL-6,iNOS and β-actin genes were established.Every assay possessed a good linear relationship between initial templates and Ct values,and the correlation coefficient of the standard curve was 0.998.It was highly sensitive and had a detection limit of 1×101 copies/μL of initial templates.It was highly specific and the fluorescent signals could only be detected by the reaction with cDNA,specific primer and probe for each gene.It was highly reproducible and had a coefficient of variation less than 2 percent for both intra-and inter-assay.The established assays were successfully used to detect IL-6 and iNOS mRNA expression levels in brain and heart tissues from mice experimentally infected with porcine encephalomyocarditis virus（EMCV） GXLC strain.The high sensitivity,specificity and reproducibility of the assays indicated that the TaqMan real-time PCR could be used as an effective tool for detection and quantification of IL-6 and iNOS.

关 键 词：小鼠 IL-6 INOS 荧光定量PCR 脑心肌炎病毒 

分 类 号：Q78[生物学—分子生物学]