逆转录—聚合酶链反应—酶联夹心杂交法定量检测人IL-6mRNA  

RT—PCR—enzyme—linked sandwich hybridization for quantification of human IL—6 mRNA ZOU Lilin, LU Jianxin

作　　者：邹立林[1] 吕建新[1] 

机构地区：[1]温州医学院检验医学与公共卫生学院,浙江温州325027

出　　处：《检验医学教育》2002年第1期43-46,共4页

摘　　要：目的:建立一种高灵敏度、高特异性、精确、简便的逆转录—聚合酶链反应—微孔板酶联夹心杂交技术定量检测人IL-6mRNA的方法。方法:从人外周静脉血单个核细胞提取总RNA,进行RT-PCR扩增,PCR产物经热变性后与检测探针一起加入已包被捕获探针的微孔板内进行夹心杂交,用链亲和素—辣根过氧化物酶(SAV—HRP)及底物系统检测杂交信号。结果:该方法检测IL-6mRNA的灵敏度明显高于逆转录—聚合酶链反应—琼脂糖凝胶电泳;特异性高,RT-PCR和酶联夹心杂交均无非特异阳性信号出现;重复性良好。结论:本方法操作简单、灵敏度高、特异性强、结果数据化,适合于细胞因子mRNA的定量检测。Objective : To develop a sensitive, specific, accurate and simple method for quantitative detection of IL—6 mRNA. Methods: Total RNA was extracted from human PBMC (peripheral blood monocytes), then amplified by RT—PCR. Heat—denatured products and detection probe were added to the wells coated with capture probe and then sandwich hybridization occurs among the capture probe, target amplicon and detection probe. The hybridization signals were detected utilizing streptavidin—peroxidase and its substract. Results: This method was more sensitive than RT—PCR—agarose gel electrophoresis, and highly specific since no non—specific signal was observed by analysis with RT—PCR and enzyme—linked sandwich hybridization. Reproducibility was also evaluated in this paper. Conclusions: With its simplicity, sensitivity, specificity and digitized results, RT—PCR—enzyme—linked sandwich hybridization can be a method of choice for assay of cytokine mRNA.

关 键 词：酶联夹心杂交法 定量检测 IL-6MRNA 逆转录-聚合酶链反应 白细胞介素-6 核糖核酸 

分 类 号：R446.61[医药卫生—诊断学]