猪促炎细胞因子多重TaqMan荧光定量RT-PCR检测方法的建立及应用  

Establishment and application of a multiplex TaqMan real-time RT-PCR assay for detecting porcine proinflammatory cytokines

作　　者：施开创[1] 梁媛[1] 陈芳芳[1] 屈素洁[1] 莫胜兰[1] 李军[1] 

机构地区：[1]广西动物疫病预防控制中心,南宁530001

出　　处：《中国农业大学学报》2012年第2期117-123,共7页Journal of China Agricultural University

基　　金：广西科学基金项目(桂科青0728047)

摘　　要：为建立及应用定量检测猪促炎细胞因子mRNA表达水平的方法,从分子水平研究脑心肌炎病毒(EMCV)的致病机制,分别构建含有猪促炎细胞因子IL-1β、IL-6、TNF-α以及管家基因β-actin基因片段的重组质粒标准品,建立了检测IL-1β/β-actin、IL-6/β-actin、TNF-α/β-actin的多重TaqMan real-time PCR检测方法。标准曲线的相关系数均达到0.998以上;初始模板的检出下限均达到10拷贝/μL;只有以目标cDNA为模板,并加入特异性引物和探针的反应才能检测到荧光信号;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株感染仔猪心肌中IL-1β、TNF-α、IL-6mRNA的表达水平进行检测。结果表明,所建立的多重TaqMan real-time PCR检测方法灵敏度高、特异性强、重复性好,可以用于猪IL-1β、IL-6、TNF-α基因的检测及定量分析。This study is to establish and apply quantitative methods for detecting the mRNA expression of porcine proinflammatory cytokine.In order to study the pathogenesis of encephalomyocarditis virus（EMCV） in molecular level,a recombinant plasmid containing the fragment of target gene,i.e.porcine IL-1β,IL-6 and TNF-α genes and housekeeping gene β-actin,were constructed as standard control.Thereafter,one multiplex real-time RT-PCR assay based on TaqMan probe for detection of IL-1β/β-actin,IL-6/β-actin,TNF-α/β-actin genes was established.The correlation coefficient of the standard curves was over 0.998;The detection limit reached 10 copies/μL of initial templates;The fluorescent signals could only be detected by the reaction with cDNA,specific primer and probe for each cytokine;The coefficient of variation was less than 2 percent for both intra-and inter-assay.The established assays were successfully used to detect IL-1β,IL-6 and TNF-α mRNA expression levels in heart tissue from piglets experimentally infected with porcine EMCV GXLC strain.The multiplex TaqMan real-time RT-PCR could be used as an effective tool for detection and quantification of these proinflammatory cytokines with high sensitivity,specificity and reproducibility.

关 键 词：猪 促炎细胞因子 脑心肌炎病毒 多重荧光定量RT-PCR TAQMAN探针 

分 类 号：Q938.15[生物学—微生物学]