猪IL-2、IL-12p40和IFN-γ SYBR Green Ⅰ real-time PCR检测方法的建立  

Development of a Real-time PCR Assay Based on SYBR Green Ⅰ for Detection of Porcine IL-2,IL-12p40 and IFN-γ

作　　者：施开创[1] 莫胜兰[1] 许瑞胜[2] 李向涛[2] 陈宏备[2] 郑敏[1] 刘棋[1] 

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

出　　处：《中国畜牧兽医》2010年第7期74-79,共6页China Animal Husbandry & Veterinary Medicine

基　　金：广西科学基金项目(桂科青0832057;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：针对猪Th1型细胞因子IL-2、IL-12p40、IFN-γ以及管家基因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-2、IL-12p40、IFN-γ及β-actin的SYBR Green Ⅰ real-ti mePCR方法。该方法线性关系好,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100拷贝/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3.5%。应用所建立的方法对猪繁殖与呼吸综合征病毒感染仔猪外周血单个核细胞中IL-2、IL-12p40和IFN-γ mRNA的表达水平进行了检测。结果表明,所建立的real-time PCR检测方法灵敏度高、特异性强、重复性好。A pair of primers was designed according to the sequence of porcine Th1-type cytokines,IL-2,IL-12p40 and IFN-γ gene as well as housekeeping gene β-actin,respectively,and a recombinant plasmid containing the target gene was constructed as a standard control for each cytokine.Then,a real-time PCR assay based on SYBR c for detection of IL-2,IL-12p40,IFN-γ and β-actin was established,respectively.Every assay had a good linear relationship between initial templates and Ct values,and the correlation coefficient of the standard curve was over 0.997.It was highly sensitive and had a detection limit of 100 copies/μL.It was highly specific and there was single specific melting peak of every cytokine.It was highly reproducible and had a coefficient of variation less than 3.5 percent for both intra-and inter-assay.The established assays were successfully used to detect IL-2,IL-12p40 and IFN-γ mRNA expression levels in peripheral blood mononuclear cells from 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 these Th1-type cytokines.

关 键 词：猪 TH1型细胞因子 荧光定量PCR SYBR Green Ⅰ 

分 类 号：Q78[生物学—分子生物学]