猪GM-CSF荧光定量PCR检测方法的建立  

Development of a Fluorescent Quantitative PCR Assay for Porcine GM-CSF

作　　者：王霞泰[1] 蒋伟娇[1] 周双海[1] 汤世坤[1] 刘凤华[1] 

机构地区：[1]北京农学院动物科学技术系,北京102206

出　　处：《北京农学院学报》2009年第2期51-53,56,共4页Journal of Beijing University of Agriculture

基　　金：国家自然科学基金资助项目(30600442);北京市自然科学基金资助项目(6082008);北京市属市管高校人才强教计划资助项目(2007)

摘　　要：根据GenBank中猪粒细胞-巨噬细胞集落刺激因子(GM-CSF)基因序列设计一对引物,用RT-PCR技术从猪外周血单个核细胞中扩增出296 bp cDNA片段,并克隆到pGEM-T Easy载体中。经测序鉴定后,构建出含猪GM-CSF cDNA部分片段的重组质粒。通过重组质粒的Real-time PCR方法,建立了猪GM-CSF cDNA的Re-al-time PCR标准曲线及其直线回归方程。该方法重复性好,敏感性高、特异性强,可用于猪GM-CSF mRNA水平的定量检测。A pair of primers for porcine granulocyte-macrophage colony-stimulating factor （GM-CSF） gene was designed according to its sequence available in GenBank,a cDNA segment of 296 base pair （bp） was amplified by RT-PCR method from porcine peripheral blood mononuclear cells, and the segment was cloned into pGEM-T Easy vector. One recombined plasmid contained the 296 bp cDNA segment was obtained after sequence analysis. The standard curve and the corresponding linear regression equation of porcine GM-CSF cDNA were obtained by real-time PCR assay. The results showed that the standard curve was of high specificity, sensitivity, and reproducibility, and provided a method to quantify porcine GM-CSF mRNA.

关 键 词：猪 GM-CSF 荧光定量PCR 

分 类 号：S852.4[农业科学—基础兽医学]