RPA Combined With CRISPR /Cas12a for Rapid and Ultrasensitive Detection Dual‐Gene of Methicillin‐Resistant Staphylococcus aureus ( MRSA )

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.