【作者】 刘金映； 张江艳； 成永强；

【Author】 Jinying Liu;J.Zhang;Y.Cheng;College of Chemistry and Environmental Science, Hebei University;

【机构】 河北大学化学与环境科学学院；

【摘要】 MicroRNA(mi RNA)是一种内源性非编码小分子RNA,影响细胞发育、凋亡、肿瘤发生等过程,因此,mi RNA的检测有重要意义。因为mi RNA自身序列较短、低表达及同族序列相似等因素,高灵敏的mi RNA检测更多依赖扩增手段。其中,核酸酶辅助循环扩增是一种利用核酸酶特异降解探针的反应,具有操作简便,灵敏度高、特异性好的优点。分子信标探针(MB)背景荧光信号低且特异性好,其与核酸酶辅助循环扩增反应结合,可用于mi RNA的检测,但是MB的茎部易被核酸酶水解,造成假阳性信号。在此,我们分别选用双链特异性核酸酶(DSN)和T7核酸外切酶(T7 EXO),通过优化MB茎部互补碱基数目,使MB探针不被核酸酶水解。当有目标mi RNA存在时,荧光标记的MB探针才可以被核酸酶水解并形成循环扩增反应,达到信号大大增强的效果。我们构建了简单灵敏、成本低、特异性好的方法,在临床疾病的诊断方面具有一定的理论指导意义和很好的应用前景。更多还原

【Abstract】 MicroRNA(miRNA) affects many diseases, especially cancer. Therefore, it is essential to establish a simple and sensitive miRNA detection method. molecular beacon probes(MB) has the advantages of detection-without separation, high sensitivity and selectivity, but the stem in the MB is easy to be hydrolized by nuclease, leading to the false positive signal. Herein, by controlling the length of the stem, the MB probe used in this method can be prevented from hydrolysis by nuclease, including duplex-specific nuclease(DSN) and T7 exonuclease(T7 Exo). Only in the presence of target miRNA is the MB probe hybridized with the target miRNA, and then hydrolized by nuclease. At the same time, the target miRNA is released and subsequently initiates the nuclease-assisted cyclic digestion process, generating enhanced fluorescence signal significantly. A simple, highly sensitive, and specific assay was developed for the homogeneous detection of miRNA by combining MB probes and nuclease-assisted cyclic amplification.更多还原