Direct visual detection for methicillin-resistant Staphylococcus aureus in milk based on the RPA-Cas12a-LFS method

Abstract

Methicillin-resistant Staphylococcus aureus (MRSA) has emerged as a significant pathogen of global concern, presenting substantial public health risks through foodborne transmission and contributing to elevated morbidity and mortality rates worldwide. The critical need for precise and timely identification of MRSA in food matrices has become increasingly paramount for effective public health protection. While numerous CRISPR-based detection platforms for MRSA have been recently developed, their widespread implementation has been hindered by intricate multi-step protocols and dependency on sophisticated instrumentation. In this study, we developed a direct, accurate and visual detection method for MRSA — the RPA-Cas12a-LFS method. This method comprises two main components: (1) the direct one-pot RPA-Cas12a system, which integrates bacterial lysis, RPA nucleic acid amplification, and CRISPR-Cas12a nucleic acid detection into a single step performed simultaneously at a constant temperature, and (2) the streptavidin-gold nanoparticles (SA-AuNP)-based CRISPR-specific lateral flow strip (LFS). By eliminating the need for nucleic acid extraction, this method significantly simplifies the experimental procedure and reduces the risk of cross-contamination. Through systematic optimization, this method demonstrated exceptional performance, enabling direct and specific identification of MRSA at remarkably low concentrations (10 CFU/mL) within 60 min in various milk samples. This advanced detection method, characterized by its direct sample processing, exceptional accuracy, visual interpretability, cost-efficiency, and minimal equipment requirements, is particularly suitable for on-site and real-time monitoring of pathogenic bacteria in the food industry.