猪IL-4、IL-6和IL-10 SYBR Green Ⅰ real-time PCR检测方法的建立及应用  

Development and application of a SYBR Green Ⅰ-based real-time PCR assay for detection of porcine IL-4, IL-6 and IL-10

作　　者：施开创[1,2] 李焕荣[1,3] 杨汉春[1] 郭鑫[1] 盖新娜[1] 

机构地区：[1]中国农业大学动物医学院／农业部人畜共患病重点开放实验室,北京100193 [2]广西动物疫病预防控制中心,广西南宁530001 [3]北京农学院动物科学技术系,北京102206

出　　处：《中国预防兽医学报》2010年第2期102-107,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家973计划项目(2005CB523204)

摘　　要：为探讨PRRSV感染后机体的体液免疫应答、从分子水平深入研究PRRSV的免疫机制,本研究针对Th2型细胞因子IL-4、IL-6、IL-10以及看家基因β-actin的基因序列分别设计一对特异性引物,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-4、IL-6、IL-10及β-actin的SYBR Green Ⅰ real-time PCR方法。该方法线性关系好,各种细胞因子及β-actin标准曲线的相关系数均达到0.997以上;敏感性高,初始模板的检出下限均达到1×101copies/μL;特异性强,扩增产物形成单一的特异性熔解峰;重复性好,组内与组间的变异系数均小于3%。应用所建立的方法对猪繁殖与呼吸综合征病毒(PRRSV)感染仔猪外周血单个核细胞(PBMC)中IL-4、IL-6和IL-10mRNA的表达水平进行了检测。结果表明,本研究建立的real-timePCR检测方法灵敏度高、特异性强、重复性好,可以用于猪Th2型细胞因子的检测及定量分析。Real-time PCR assays based on SYBR Green I for detection of IL-4, IL-6, IL-10 and 13-actin were established us- ing primers derived from porcine Th2-type cytokines （IL-4, IL-6 and IL-10） gene. The assays were highly sensitive and had a detection limit of 1 × 10^1 copies/μL of initial templates. These assays were highly specific and there was single specific melting peak for every cytokine. It was highly reproducible and had a coefficient of variation less than 3 percent for both intra-and inter-assay. The established assays were successfully used to detect IL-4, IL-6 and IL-10 mRNA expression levels in peripheral blood mononuclear cells （PBMCs） in piglets experimentally infected with porcine reproductive and respiratory syndrome virus （PRRSV）. The high sensitivity, specificity and reproducibility of the assays indicated that the SYBR Green I real-time PCR could be used as an effective tool for detection and quantification of Th2-type cytokines.

关 键 词：猪 TH2型细胞因子 荧光定量PCR 检测方法 

分 类 号：S852.65[农业科学—基础兽医学]