Simple Isothermal Strategy for Multiplexed, Rapid, Sensitive, and Accurate miRNA Detection
Simple Isothermal Strategy for Multiplexed, Rapid, Sensitive, and Accurate miRNA Detection
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DOI：
10.1021/acssensors.6b00105
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发表时间：
2016-06-01
期刊：
影响因子：
8.9
通讯作者：
Trau, Matt
中科院分区：
文献类型：
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作者：
Wee, Eugene J. H.;Trau, Matt
microRNA（miRNA）是潜在的生物标志物。目前优选的miRNA检测方法依赖于各种基于PCR的方法。虽然有效，但这些方法通常是繁琐、缓慢的，并且需要昂贵的设备。因此，仍然需要更快和更简单的miRNA检测方法。在此，我们描述miRPA：重组酶聚合酶扩增（RPA）与PBCV-1 DNA连接酶的新型组合用于简单、特异、快速和多重等温miRNA检测。MiRPA对皮克水平的总RNA输入（或类似于40拷贝/pg）敏感，并且可以区分密切相关的miRNA。MiRPA应用于细胞系，随后使用商业qPCR方法进行验证。通过检测尿源性RNA中的miRNAs也证明了潜在的临床应用。这是RPA首次应用于快速等温miRNA检测，它作为miRNA传感器在研究和临床中具有广泛的应用。
MicroRNAs (miRNA) are potential biomarkers. Current preferred miRNA detection methods rely on various PCR-based approaches. Although effective, such methods are typically tedious, slow, and require expensive equipment. Hence, faster and simpler miRNA detection approaches are still needed. Herein, we describe miRPA: a novel combination of recombinase polymerase amplification (RPA) with PBCV-1 DNA ligase for simple, specific, rapid, and multiplexed isothermal miRNA detection. MiRPA is sensitive to picogram levels of total RNA input (or similar to 40 copies/pg) and can discriminate between closely related miRNAs. MiRPA was applied to cell lines and was subsequently validated with a commercial qPCR method. Potential clinical application was also demonstrated by detecting miRNAs in urine-derived RNA. This is the first application of RPA for rapid isothermal miRNA detection, and it could have wide applications as a miRNA sensor in both research and in the clinic.