Carbapenem-resistant Klebsiella pneumoniae (CRKP) poses a major public health threat due to high mortality and limited therapeutic options. Rapid and accurate detection of both species identity and resistance determinants is critical for clinical management. Here, we developed a dual-target recombinase polymerase amplification-CRISPR/Cas12a multiplex lateral flow assay (DRC-MLFA) for simultaneous detection of K. pneumoniae and blaKPC in a single reaction. This platform introduces a functionally differentiated dual-channel detection strategy, in which the K. pneumoniae signal is generated directly via RPA-lateral flow readout, while blaKPC detection is achieved through an additional CRISPR/Cas12a-mediated verification step. To enable one-pot integration, suboptimal protospacer adjacent motif (PAM) sequences were employed to delay Cas12a activation, thereby coordinating amplification and CRISPR detection within a single reaction system. Under optimized conditions, the assay achieved dual-target detection within 50 min at 37 °C, with a limit of detection of 101 CFU/μL and high specificity against non-target pathogens. Validation with 30 clinical samples showed strong concordance with quantitative PCR. This portable and low-cost biosensor provides a practical solution for point-of-care detection of CRKP and establishes a framework for integrating hierarchical specificity control into multiplex CRISPR diagnostics.
