酶标板上基于竞争杂交反应的几种miRNA序列的比色检测

多种miRNA序列的同时检测对癌症的预警与早期诊断具有重要的意义。本文基于生物素标记的miRNA和靶点miRNA与酶标板上固定的寡核苷酸探针之间的竞争反应,通过比色法来定量检测三种与胶质瘤相关的miRNA序列,即miR-182、miR-185和miR-381。通过亲和素与生物素之间的特异性相互作用,将亲和素标记的多聚辣根过氧化物酶(SA-polyHRP)衍生到微孔表面。通过检测HRP催化3,3′,5,5′-四甲基联苯胺和H2O2的显色反应来对三种靶点miRNA序列进行定量。方法最低检测浓度为10fmol/L,有望用于临床上miRNA的定量分析。碱基错配的结果表明该方法具有较好的选择性。

Colorimetric method for multiplexed miRNA detection on ELISA plates based on competitive hybridization reaction

Multiplexed miRNA detection is of great importance for early diagnosis of cancers. In this study, a colorimetric method based on the competitive hybridization reaction has been developed in which the biotinylated miRNA （biotin-miRNA） and target miRNA compete for the immobilized oligonucleotide （ODN） probe. Targets miR-182, miR-185 and miR-381 were analyzed using the present method. Relying on the specific interaction between avidin and biotin, complex of avidin-poly-horseradish peroxidase （SA-poly-HRP） was introduced onto the wells of the ELISA plates. The incorporated HRP could catalyze the chromogenic reac- tion of 3, 3＇, 5, 5＇-tetramethylbenzidine and Hz 02. The method is highly selective, and could determine target miRNAs with concentrations as low as 10 fmol/L, thus holding great potential for clinical miRNA assay.