应用竞争PCR定量检测猪IFN-γ mRNA  

Quantitative Detection of Porcine IFN-γ mRNA by Competitive PCR

作　　者：郭海龙[1] 童光志[1] 仇华吉[1] 周艳君[1] 兰文生[1] 王云峰[1] 吴东来[1] 

机构地区：[1]中国农业科学院哈尔滨兽医研究所兽医生物技术国家重点实验室,黑龙江哈尔滨150001

出　　处：《兽医大学学报》2003年第3期278-280,共3页

基　　金：国家重点基础研究发展规划 ( 973 )资助项目( G19990 1190 2 )

摘　　要：运用 RT- PCR从经 Con A体外刺激培养的猪外周血单核细胞 (PBMC)提取的细胞总 RNA中扩增猪 γ-干扰素(IFN-γ)的部分片段 ,经酶切鉴定后双酶切缺失中间一小片段 ,将余下的 2个较大片段连接 ,并用相同的引物在相同条件下扩增连接产物 ,从而获得截短的同源性片段。将其纯化后克隆于 p MD1 8- T载体 ,重组质粒经酶切和 PCR鉴定后进行序列测定 ,验证无误后作为竞争性模板内对照 ,一定比例稀释后分别与等体积经刺激培养的 PBMC总 RNA反转录产物进行 PCR,产物电泳后经扫描分析获得各目标条带与竞争条带的溴化乙锭 (EB)嵌入光密度值 (IOD) ,经校正后取上述两者比值的对数值作为 Y轴 ,取已知竞争模板相应浓度的对数值作为 X轴 ,绘制成竞争曲线。应用建立的竞争 PCR对不同样品重复试验表明 ,本方法操作简单 ,结果可靠 ,稳定性高 ,适用于猪 IFN-γ mThe total RNA was extracted from ConA stimulated porcine peripheral blood mononuclear cell(PBMC) and used for reverse transcription.Partial fragment of porcine IFN γ was amplified from the cDNA by PCR and was further deleted a small part in the middle region by a double digestion.The shorten fragmens were religated together and amplified by PCR using the same primers,and then cloned into pMD18 T vector.After identification,the positive clones were sequenced and used as competitive templates.The diluted templates and the cDNA were co amplified in the same tubes,and the PCR products were electrophoresed and scanned,the integrated optical density(IOD) of the targeted and competitive DNA bands were detected,and a competitive curve was drawn.Using the developed competitive PCR for detection of porcine IFN γ mRNA showed that the assay is simple and repeatable.

关 键 词：竞争PCR 定量检测 猪 IFN-γmRNA 序列测定 

分 类 号：S828[农业科学—畜牧学] Q754[农业科学—畜牧兽医]