Rapid and sensitive detection of blaKPC and blaNDM in carbapenemase-producing Enterobacterales using a one-pot recombinase polymerase amplification–CRISPR/Cas12a system with modified protospacer adjacent motifs

Carbapenem-resistant Enterobacterales pose a critical global health threat because of their rapid transmission and resistance to last-line antibiotics. To address this threat, rapid and user-friendly point-of-care (POC) detection is essential. Quantitative polymerase chain reaction (qPCR), while providing high sensitivity, relies on expensive equipment and skilled personnel, hindering its practical use in resource-limited settings. Here, we aimed to develop an optimized one-pot RPA–CRISPR/Cas12a (RCCS) system with enhanced diagnostic performance to provide a reliable and user-friendly platform for rapid carbapenemase gene screening in both clinical and resource-limited settings. The developed assay used for the detection of bla KPC and bla NDM genes, which was integrated into a portable diagnostic device. By using suboptimal protospacer adjacent motif sequences, the assay provided a streamlined workflow and substantially enhanced detection sensitivity. This platform achieved a limit of detection of 10 -17 M for bla KPC and 10 -16 M for bla NDM within 30 min. Validation with 44 clinical samples demonstrated that the assay had 100% sensitivity and specificity, matching the effectiveness of qPCR. The one-pot RCCS platform offers a robust and highly-sensitive POC solution for on-site testing.