The use of microRNAs as clinical cancer biomarkers is hindered by the absence of accurate, fast and inexpensive assays for their detection in biofluids. Here we report a one-step and one-pot isothermal assay that leverages rolling-circle amplification and the endonuclease Cas12a for the accurate detection of specific miRNAs. The assay exploits the cis-cleavage activity of Cas12a to enable exponential rolling-circle amplification of target sequences and its trans-cleavage activity for their detection and for signal amplification. In plasma from patients with pancreatic ductal adenocarcinoma, the assay detected the miRNAs miR-21, miR-196a, miR-451a and miR-1246 in extracellular vesicles at single-digit femtomolar concentrations with single-nucleotide specificity. The assay is rapid (sample-to-answer times ranged from 20 min to 3 h), does not require specialized instrumentation and is compatible with a smartphone-based fluorescence detection and with the lateral-flow format for visual readouts. Simple assays for the detection of miRNAs in blood may aid the development of miRNAs as biomarkers for the diagnosis and prognosis of cancers.

由于缺乏准确、快速和廉价的生物流体检测方法，因此阻碍了 microRNA 作为临床癌症生物标志物的使用。在这里，我们报道了一种一步法和一锅法等温测定法，该方法利用滚环扩增和核酸内切酶 Cas12a 来准确检测特异性 miRNA。该检测利用 Cas12a 的顺式切割活性来实现靶序列的指数滚环扩增及其反式切割活性，用于检测和信号放大。在胰腺导管腺癌患者的血浆中，该测定法在细胞外囊泡中检测到个位数飞摩尔浓度的 miRNAs miR-21 、 miR-196a 、 miR-451a 和 miR-1246 具有单核苷酸特异性。该检测速度快（从样品到结果的时间从 20 分钟到 3 小时不等），不需要专门的仪器，并且与基于智能手机的荧光检测和用于视觉读数的横流格式兼容。检测血液中 miRNA 的简单检测可能有助于开发 miRNA 作为癌症诊断和预后的生物标志物。