Rapid and accurate detection of methicillin-resistant Staphylococcus aureus (MRSA) is crucial for guiding antimicrobial therapy and improving outcomes in infection management, including postoperative care and surgical site infections. Conventional detection methods often suffer from lengthy turnaround times, operational complexity, or limited specificity. To address these challenges, we developed a novel, integrated biosensing platform termed the Cas12a/crRNA-initiated triple self-priming cascade amplification assay for ultrasensitive and specific analysis of the mecA resistance gene. The innovation of this strategy lies in the synergistic combination of CRISPR-Cas12a for high-fidelity target recognition and an elegantly engineered isothermal amplification circuit. Target-specific activation of Cas12a triggers the trans-cleavage of a designed probe, remaining an RNA initiator that sets off a triple self-priming cascade amplification within a single, multifunctional Y-shaped probe (Y-probe). This integrated probe architecture eliminates the need for multiple independent components, significantly simplifying the workflow and enhancing assay stability. Amplification culminates in the generation of G-quadruplex (G4) structures, which are detected through a label-free turn-on fluorescence signal using thioflavin T (ThT), thereby avoiding the instability associated with covalently labeled probes. The assay demonstrated exceptional analytical performance, achieving a detection limit of 0.41 fM for synthetic mecA DNA and 5.2 CFU/mL for viable MRSA cells, with a linear range spanning five to six orders of magnitude. It exhibited high specificity, successfully discriminating MRSA from methicillin-susceptible Staphylococcus aureus and distinguishing single-base mismatches. By integrating precise CRISPR recognition, a streamlined and stable all-in-one probe design, and a robust label-free readout, the platform presents a powerful, user-friendly molecular tool for resistance genotyping, with potential for future application in clinical settings, such as postoperative infection management.
