Method Development and Sensitivity Optimization Paradigm for One-Step RPA-CRISPR/Cas12a Rapid Detection of the Hard-to-Amplify Gene <i>bla</i> _NDM

The presence and spread of antibiotic resistance genes (ARGs) across various habitats have increased the risks of antibiotic resistance, highlighting the urgent need for effective monitoring methods. One key challenge in method development lies in balancing sensitivity, speed, and portability. To address it, a one-step assay targeting the carbapenem resistance gene <i>bla</i>_NDM was developed based on recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a. A sensitivity optimization paradigm─MOSAIC (multistrategy optimized sensitive assay via integrated CRISPR/Cas12a)─was proposed, incorporating component optimization, suboptimal-PAM-mediated CRISPR inhibition, and glycerol-assisted phase separation. The glycerol-assisted strategy exhibited the largest enhancement, followed by the suboptimal-PAM strategy and component optimization. When combined, these strategies demonstrated a synergistic effect, yielding greater improvement (10 000-fold) than a single strategy alone. MOSAIC reached a limit of detection (LOD) of 260 copies/μL, comparable to that of qPCR, and enabled faster quantification of <i>bla</i>_NDM at 37 °C within 1 h on a standard plate reader. It achieved 100% diagnostic sensitivity and 95.45% specificity in clinical isolates, and 77.41-99.73% accuracy in environmental matrix-spiked samples, comparable to that of qPCR. It provides a technological foundation for on-site detection of <i>bla</i>_NDM and offers an optimization paradigm and new insights for the development of one-step RPA-CRISPR/Cas12a assays targeting various genes.