小鼠IL-1β、TNF-αTaqMan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

摘要

In this study, a real-time RT-PCR assay based on TaqMan probe for detection of mouse proinflammatory cytokine gene IL-1 p 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-1 P and TNF-a 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.%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10 copies/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-1 β、TNF-α mRNA的转录水平进行检测,发现感染后第4d IL-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.