小鼠IL-1β、TNF-α TaqMan荧光定量RT-PCR检测方法的建立及脑心肌炎病毒感染小鼠的检测  

Development of a TaqMan real-time PCR assay for detection of IL-1β and TNF-α mRNA in mice experimentally infected with encephalomyocarditis virus

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

机构地区：[1]广西大学动物科学技术学院,广西南宁530005 [2]广西动物疫病预防控制中心,广西南宁530001

出　　处：《中国预防兽医学报》2011年第7期531-536,共6页Chinese Journal of Preventive 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)感染后促炎细胞因子的表达水平、从分子水平深入研究EMCV的致病机制,本研究分别建立了检测小鼠IL-1β、TNF-α和管家基因β-actin 的 TaqMan real-time PCR 检测方法。该方法标准曲线的相关系数均达到0.998以上,检出下限均达到10copies/μL质粒标准品,组内与组间的变异系数均小于2%。应用该方法对猪源 EMCV GXLC 株人工感染小鼠的脑、心、脾中IL-1β、TNF-α mRNA 的转录水平进行检测,发现感染后第4dIL-1β、TNF-α mRNA 的转录水平达到峰值,并且与小鼠发病死亡高峰存在明显的时间相关性。本研究所建立的TaqMan real-time PCR 检测方法为小鼠促炎细胞因子的检测及定量分析提供了技术手段。In this study, a real-time RT-PCR assay based on TaqMan probe for detection of mouse proinflammatory cytokine gene IL-1β and TNF-α was established, respectively. The assays were highly specific, sensitive and reproducible, of which the correlation coefficient of the standard curve was over 0.998, the sensitivity was 10 copies/μL of standard recombinant plasmid and the coefficient of variation was less than 2 percent for both intra-assay and inter-assay. The established assays were used to detect IL-1β and TNF-α mRNA levels in brain, heart and spleen tissues of mice experimentally infected with porcine encephalomyocarditis virus （EMCV） GXLC strain. The results showed that IL-1β and TNF-α mRNA expression levels reached peak value at 4 day post EMCV infection, with a time correlation between the expression levels and the mortality of infected mice. The results indicated that the TaqMan real-time PCR assay could be used as an effective tool for detection and quantification of these proinflammatory cytokines.

关 键 词：脑心肌炎病毒 小鼠 促炎细胞因子 荧光定量RT-PCR 

分 类 号：S852.72[农业科学—基础兽医学]