结合恒温滚环扩增(rolling circle amlification,RCA)、单链特异性核酸外切酶工(exonuclease Ⅰ,Exo Ⅰ)和阳离子共轭聚合物(Cationic conjugated polymer,CCP)荧光共振能量转移(fluorescence resonance energy transfer,FRET)技术,建立了一种特异、灵敏的均相检测microRNA(miRNA)的新方法.该方法应用荧光标记探针与RCA扩增的长链DNA产物杂交,当加入CCP时,其与杂交的标记探针通过静电力结合,发生高效的FRET.未杂交的标记探针利用Exo Ⅰ水解成单核苷酸,其与CCP相互作用力弱,不能发生有效的FRET.基于此,无需分离和洗涤步骤,实现了RCA扩增miRNA的均相检测.方法特异性好,灵敏度高,线性为0.5～20 pmol/L,检出限为0.2 pmol/L.方法为miRNA均相检测和原位成像分析以及临床诊断提供了新策略.
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