Zeptomolar-level one-pot simultaneous detection of multiple colorectal cancer microRNAs by cascade isothermal amplification

Abstract

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.

Keywords: Cascade isothermal amplification; Colorectal cancer; One-pot multiple microRNA detection.