微量酶标夹心杂交法定量检测人iNOS-mRNA  

An enzyme—linked sandwich hybridization on microplate for quantification of human iNOS mRNA

作　　者：许艳芳[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细胞与分子医学研究所,浙江温州325027

出　　处：《检验医学教育》2003年第1期36-39,共4页

摘　　要：目的:建立一种高灵敏度、高特异性、精确、简便的微孔板酶联夹心杂交技术,对人iNOS—mRNA进行定量检测。方法:设计两条特异探针,其中一条为用捕获探针,5’端连接氨基,能与微孔板表面的NOS基团共价结合,"竖直"地包被在微孔中,另一个为检测探针,3’端带有生物素标记。用脂多糖(LPS)刺激人外周血单个核细胞24h后,提取巨噬细胞总RNA,进行RT—PCR扩增,PCR产物热变性后加入到已包被捕获探针的微孔板内,并加入检测探针进行夹心杂交,最后加入链亲和素—辣根过氧化物酶(SAV—HRP)及底物,在450nm波长检测杂交信号。结果:该方法检测iNOS—mRNA的灵敏度明显高于RT—PCR—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iNOS—mRNA的定量检测。objective: To develop a sensitive, specific, accurate and simple method for quantitative detection of iNOS—mRNA. In peripheral blood macrophage of homo sapiens. Methods: Total RNA was extracted from human macrophage, then amplified by RT—PCR. Two probes were designed, one is capture probe; the other is detection probe. The capture probe, with amino modified at 5'—end, can couple with the N—oxysuccininide esters(NOS) group on the DNA—Binding wells through covalent attachment. In this way, oligonucleotides are immobilized on the plate surface and arise straightly to capture target amplicon. Heatdenatured products and detection probe with biotinylated at 3'—end, were then added to wells and hybridization occurs between capture probe, target amplicon and detection probe. After the addition of Streptavidin—Peroxidase developing system, the optical density were read at 450nm. 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. Conelutions: With its simplicity, sensitivity, specificity and digitized results, RT—PCR—enzyme—linked sandwich hybridization can be a method of choice for assay of iNOS—mRNA.

关 键 词：微孔板酶联夹心杂交技术 定量检测 诱导性一氧化氮合成酶 RT—PCR 分子生物学检测 

分 类 号：R446[医药卫生—诊断学]