Abstract
The mecA gene confers methicillin resistance in both MRSA and MR-CoNS by encoding the PBP2a protein and poses a significant public health threat due to its resistance to beta-lactam antibiotics. Rapid and accurate detection of mecA is critical for timely treatment, reducing morbidity, and preventing its spread in healthcare settings. In this study, we developed an advanced one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a system, enhanced with glycerol and betaine, for ultrasensitive detection of the mecA gene. Glycerol's viscosity effect prevents premature interaction between Cas12a and early amplification products, while betaine enhances nucleic acid amplification. The assay demonstrated superior sensitivity, detecting as low as 5 copies/μL of mecA DNA within 60 min. Specificity testing against a panel of bacterial species confirmed the high selectivity of the assay for mecA-carrying strains with negligible cross-reactivity. Furthermore, this method exhibited excellent performance across various clinical samples, including blood, urine, and bronchoalveolar lavage fluid. Our findings underscore the potential of this advanced RPA-CRISPR/Cas12a assay as a powerful diagnostic tool for rapid, cost-effective, and highly sensitive mecA detection, offering a promising solution for clinical diagnostics and infection control.