Naked-Eye and On-Site Detection of Staphylococcus aureus via DNAzyme-Assisted Colorimetric Bioassay for CRISPR/Cas12a

Dairy products have become a key part of the population's diet due to their nutritional richness, but with increasing demand and market expansion, concerns about the quality and safety of these products have intensified, drawing public attention to the potential risks involved. Their nutritional properties are conducive to the growth of microorganisms, which can lead to contamination with pathogenic bacteria and foodborne diseases. As a common foodborne pathogen, <i>Staphylococcus aureus</i> is one of the main causes of foodborne diseases worldwide, and it is also a major source of the contamination of dairy products, posing a serious threat to human health. Although the traditional microbial culture method is accurate, it is cumbersome and time-consuming and requires a sterile environment and large-scale equipment, making it difficult to detect bacteria rapidly. Therefore, the development of convenient, accurate, and sensitive on-site detection methods is essential. In this study, we combined CRISPR/Cas12a technology and DNAzyme colorimetric signal output to design a naked-eye output instant detection platform for <i>S. aureus</i>, which can realize the naked-eye reading of the test results. After system optimization, our detection method achieved a detection limit of 10⁰ CFU/mL for pure <i>Staphylococcus aureus</i> culture, with a linear range of 10⁰-10⁸ CFU/mL (R² = 0.908). This method exhibits good specificity and can accurately identify <i>Staphylococcus aureus</i> and other common foodborne bacteria (<i>Salmonella</i>, <i>Escherichia coli</i>, <i>Listeria monocytogenes</i>, <i>Lactobacillus plantarum</i>). It is crucial that when applied to artificially contaminated milk and milk beverages, this method still maintains a detection limit of 10⁰ CFU/mL, demonstrating its strong performance in complex food matrices without the need for complex DNA extraction. This CRISPR/Cas12a DNAzyme colorimetric bioassay can quickly (<2 h) and visually interpret results, providing a powerful, low-cost, and field-deployable tool for enhancing food safety monitoring.