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基于三链结构触发辅助指数酶扩增的 miRNA 的超灵敏和快速检测。

Ultrasensitive and rapid detection of miRNA with three-way junction structure-based trigger-assisted exponential enzymatic amplification.

机构信息

Key Laboratory of Chemical Sensing & Analysis in Universities of Shandong, College of Biological Sciences and Technology, University of Jinan, Jinan 250022, PR China.

College of Resources and Environment, University of Jinan, Jinan 250022, PR China.

出版信息

Biosens Bioelectron. 2016 Jul 15;81:236-241. doi: 10.1016/j.bios.2016.02.034. Epub 2016 Mar 2.

Abstract

Aberrant expression of micro RNA (miRNA) is associated with development of cancers and diseases, so miRNA has become a tissue-based biomarker for cancer prognosis and diagnosis. Herein, a novel trigger-assisted exponential enzymatic amplification (T-EXPEA) method for ultrasensitive miRNA detection based on three-way junction (3-WJ) structure driven has been reported, which can be used in potential applications in cancer prognosis and diagnoses. In this assay, target miRNA can unfold hairpin probe and start the reaction, and thus specifically form stable 3-WJ structure with helper. Then it can produce triggers under the synergetic polymerase and restriction endonucleases amplification. The produced triggers could be used to unfold molecular beacon (MB) and initiate T-EXPEA process. In the EXPEA part, the exponential triggers were generated to initiate new T-EXPEA and high enhancement fluorescence amplification efficiency was obtained. The feature of our strategy lies in the T-EXPEA combining with 3-WJ structure has been utilized for fluorescence miRNA detection. It is worth noting that the sequence of the triggers in T-EXPEA part is the same to that of triggers generated from the 3-WJ part. In addition, the design of restriction enzyme cutting sites using the same restriction enzyme (Nt.BbvCI) in hairpin probe and MB respectively, improved reaction efficiency cost-efficiently. This method can quantitatively detect sequence-specific miRNA in a dynamic range from 10 aM to 10 pM with a detection limit as low as 7.8 aM.

摘要

微 RNA (miRNA) 的异常表达与癌症和疾病的发生有关,因此 miRNA 已成为癌症预后和诊断的基于组织的生物标志物。在此,报道了一种基于三链体(3-WJ)结构驱动的新型触发辅助指数酶扩增(T-EXPEA)方法,用于超灵敏 miRNA 检测,可用于癌症预后和诊断的潜在应用。在该测定中,靶 miRNA 可以展开发夹探针并开始反应,从而与辅助物特异性地形成稳定的 3-WJ 结构。然后,它可以在协同聚合酶和限制内切酶扩增下产生触发物。产生的触发物可用于展开分子信标(MB)并启动 T-EXPEA 过程。在 EXPEA 部分,指数触发物被生成以启动新的 T-EXPEA,并获得高的增强荧光扩增效率。该策略的特点在于结合 3-WJ 结构的 T-EXPEA 已用于荧光 miRNA 检测。值得注意的是,T-EXPEA 部分中的触发物序列与 3-WJ 部分中生成的触发物序列相同。此外,使用相同的内切酶(Nt.BbvCI)分别在发夹探针和 MB 中设计内切酶切割位点,提高了反应效率,降低了成本。该方法可以在 10 aM 至 10 pM 的动态范围内定量检测序列特异性 miRNA,检测限低至 7.8 aM。

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