Multi-microRNA (miRNA) detection would greatly facilitate early diagnosis of colorectal cancer (CRC). Here a convenient cascade isothermal amplification approach incorporating a G-quadruplex molecular beacon (G4MB) was established for achieving one-pot detection of multiple CRC miRNAs (miRNA-21, miRNA-92a, miRNA-31); this strategy incorporated a Bsu DNA polymerase (Bsu pol)-induced strand-displacement reaction and a Lambda exonuclease (λexo)-aided recycling reaction. In the presence of target miRNA, the G-rich stem structure was opened and became available for hybridization with the primer to initiate synthesis of Bsu pol-catalyzed double-stranded DNA (dsDNA) that displaced the miRNA target and released it, allowing it to participate in subsequent amplification cycles. Meanwhile, the dsDNA was gradually digested into fragments by λexo from the 5′ phosphorylated end, releasing the newly synthesized DNA strand for participation in subsequent cycles that led to amplification of the fluorescent signal. This approach provided a low limit of detection (LOD) of zeptomolar-level, 85.8 zM, 77.6 zM, 78.9 zM for miRNA-21, miRNA-92a, miRNA-31, respectively. It could distinguish the mismatched targets and achieved three miRNA targets detection run in parallel in one-pot within 2 h. Thus, this fast, simple, and convenient strategy holds great promise as a clinical application for the detection of multiple miRNAs in clinical CRC samples.

多微RNA（miRNA）检测将大大促进大肠癌（CRC）的早期诊断。在这里，建立了一个方便的级联等温扩增方法，该方法结合了G-四链体分子信标（G4MB），可实现一锅式检测多个CRC miRNA（miRNA-21，miRNA-92a，miRNA-31）。该策略结合了Bsu DNA聚合酶（Bsu pol）诱导的链置换反应和Lambda核酸外切酶（λexo）辅助的回收反应。在靶标miRNA存在下，富含G的茎结构被打开，可用于与引物杂交以启动Bsu的合成pol催化的双链DNA（dsDNA）取代了miRNA靶并释放了它，使其可以参与后续的扩增循环。同时，dsDNA被λexo从5'磷酸化末端逐渐消化成片段，释放出新合成的DNA链以参与随后的循环，从而导致荧光信号的扩增。此方法分别为miRNA-21，miRNA-92a和miRNA-31提供了低分子水平的检出限（LOD），分别为85.8 zM，77.6 zM和78.9 zM。它可以区分不匹配的靶标，并在2 h内在一锅中平行运行三个miRNA靶标。因此，这种快速，简单和方便的策略作为检测临床CRC样品中多种miRNA的临床应用具有广阔的前景。