SDA-mediated high-sensitivity visual CRISPR/Cas12a detection platform for point-of-care detection of MRSA

Methicillin-resistant Staphylococcus aureus (MRSA) infections progress rapidly, posing a significant threat to patient health. Culture-based drug susceptibility testing and nucleic acid amplification methods are time-consuming and complex, requiring specialized instruments and technicians. To address these limitations, we present a novel, visual POCT platform for MRSA detection with the naked eye. This visualization method uses CRISPR/ Ca s12a to accurately identify target genes, cleave the strand displacement amplification ( SD A) template, and regulate the aggregation of DNA-functionalized gold nanoparticles ( Au NPs), with the resulting color change serving as a signal (CaSD@Au). The presence of the target gene activates CRISPR/Cas12a enzymatic activity, which cleaves the SDA template. This cleavage suppresses the SDA reaction, prevents DNA-AuNP aggregation, and maintains nanoparticle dispersion, resulting in a visible red color. In the absence of the target gene, the SDA reaction proceeds, and the gold nanoparticles aggregate, turning purple. Sensitivity analysis confirmed that CaSD@Au detects MRSA with a limit of detection of 5 cfu mL −1 . When combined with recombinase polymerase amplification (RPA), CaSD@Au showed detection performance comparable to qPCR in the actual detection of 20 clinical strains. Furthermore, this platform enables results to be determined simply by observing color changes with the naked eye, without the need for complex instruments. The CaSD@Au platform offers reliable, sensitive, and specific detection, enhancing the applicability of CRISPR/Cas12a-based assays for pathogen detection, especially in resource-limited settings.