Abstract

The increasing issue of infections caused by methicillin‐resistant Staphylococcus aureus (MRSA) necessitates rapid and reliable diagnostic methods. While existing RPA‐CRISPR/Cas12a platforms have demonstrated potential for MRSA detection, most rely on single‐gene targets or require multiple Cas enzymes. Here, we have developed a novel dual gene detection strategy that simultaneously detects the S. aureus specific femA gene and the methicillin‐resistant mecA gene in a single RPA‐CRISPR/Cas12a reaction. This integrated approach enables clear discrimination between MRSA and methicillin‐sensitive Staphylococcus aureus (MSSA) in just 30 min, with results visualized via both fluorescence and lateral flow strips. The assay exhibited high specificity (no cross‐reactivity with common pathogens) and a sensitivity of 10 copies/μL, comparable to qPCR. Validation with 39 clinical samples showed 100% concordance with antimicrobial susceptibility testing. Our dual‐gene RPA‐CRISPR/Cas12a platform represents a significant advancement in point‐of‐care MRSA diagnostics, offering enhanced accuracy and operational simplicity.