Multiplex visual nucleic acid detection via isothermal cascaded transcription of fluorescent RNA aptamers

Abstract Cascade transcription amplification holds great promise for nucleic acid detection, yet achieving multiplexed visual detection with high sensitivity remains challenging. Herein, a visual platform termed Multiplexed Fluorescent‐Aptamer Generation via Isothermal Cascaded amplification (MAGIC) is presented for multiplex nucleic acid detection. MAGIC platform integrates target‐triggered exponential amplification via nicking and strand displacement with cell‐free transcription of fluorescent RNA aptamers. Target recognition triggers this exponential amplification, yielding abundant aptamer‐encoding DNA templates that are subsequently transcribed into functional fluorescent RNA aptamers. Multiplexed visual nucleic acid detection is achieved by generating multiple fluorescent aptamers with spectrally distinct fluorescence signals. MAGIC is a robust system that enables early diagnosis of drug‐resistant bacteria and achieves attomolar (10 aM) sensitivity for drug‐resistant genes. In clinical isolates, MAGIC visually diagnoses carbapenem‐resistant hypervirulent Klebsiella pneumoniae strains with 100% accuracy, demonstrating its potential for early pathogen diagnosis and point‐of‐care molecular testing.