绵羊IFN-γ和IL-2基因SYBR GreenⅠreal-time PCR检测方法的建立及应用  

Development and application of SYBR Green Ⅰ real-time PCR assays for detection of sheep IFN-γ and IL-2 genes

作　　者：张冰冰[1] 赵魁[1] 宋德光[1] 臧德跃[1] 王栋[1] 贺文琦[1] 陈克研[1] 王改丽[1] 高丰[1] 

机构地区：[1]吉林大学畜牧兽医学院,吉林长春130062

出　　处：《中国兽医科学》2010年第11期1146-1150,共5页Chinese Veterinary Science

基　　金：吉林省科技发展计划重点资助项目(20080217)

摘　　要：为探讨传染性脓疱病毒感染后机体的细胞免疫应答,从分子水平对传染性脓疱病毒的免疫机制进行了深入探讨。首先针对Th1型细胞因子IFN-γ、IL-2以及管家基因GAPDH的mRNA序列分别设计了1对特异性引物,扩增相应的基因片段并构建含有相应基因序列的重组质粒,以所构建的重组质粒作为阳性标准品,建立了用于IFN-γ、IL-2及GAPDH检测的SYBR GreenⅠreal-time PCR方法。该方法线性关系良好,IFN-γ、IL-2和GAPDH标准曲线的相关系数均达0.99以上;敏感性高,初始模板的检出下限均达1×103copies/μL;特异性强,扩增产物形成单一的特异性融解峰;重复性好,组内与组间的变异系数均小于3%。应用该方法对传染性脓疱病毒感染羔羊外周血白细胞中的IFN-γ、IL-2 mRNA的表达水平进行了检测。结果表明,建立的real-time PCR检测方法具有灵敏度高、特异性强、重复性好等优点,可用于绵羊Th1型细胞因子的检测及定量分析。Real-time PCR assays based on SYBR GreenⅠfor detection of IFN-γ,IL-2 and GAPDH genes were established using primers derived from sheep Th1-type cytokine（IFN-γ,IL-2） genes.The assays were highly sensitive and had a detection limit of 1×10 3copies/μL of initial templates.These assays were highly specific and there was single specific melting peak for each gene.They were highly reproducible and had a coefficient of variation less than 3 percent for intra-and inter-assays.The established assays were successfully used to detect IFN-γ and IL-2 mRNA expression levels in peripheral blood leukocytes in lamb experimentally infected with orf virus.The high sensitivity,specificity and good reproducibility indicated that the developed SYBR Green Ⅰreal-time PCR assays were effective tools for the detection and quantification of sheep Th1-type cytokines.

关 键 词：羔羊 TH1型细胞因子 荧光定量聚合酶链反应 检测方法 

分 类 号：S852.659.1[农业科学—基础兽医学]