Isothermal Amplification and CRISPR/Cas12a-System-Based Assay for Rapid, Sensitive and Visual Detection of Staphylococcus aureus.

Abstract

Staphylococcus aureus exists widely in the natural environment and is one of the main food-borne pathogenic microorganisms causing human bacteremia. For safe food management, a rapid, high-specificity, sensitive method for the detection of S. aureus should be developed. In this study, a platform for detecting S. aureus (nuc gene) based on isothermal amplification (loop-mediated isothermal amplification-LAMP, recombinase polymerase amplification-RPA) and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas12a) proteins system (LAMP, RPA-CRISPR/Cas12a) was proposed. In this study, the LAMP, RPA-CRISPR/Cas12a detection platform and immunochromatographic test strip (ICS) were combined to achieve a low-cost, simple and visualized detection of S. aureus. The limit of visual detection was 57.8 fg/µL of nuc DNA and 6.7 × 102 CFU/mL of bacteria. Moreover, the platform could be combined with fluorescence detection, namely LAMP, RPA-CRISPR/Cas12a-flu, to establish a rapid and highly sensitive method for the detection of S. aureus. The limit of fluorescence detection was 5.78 fg/µL of genomic DNA and 67 CFU/mL of S. aureus. In addition, this detection platform can detect S. aureus in dairy products, and the detection time was ~40 min. Consequently, the isothermal amplification CRISPR/Cas12a platform is a useful tool for the rapid and sensitive detection of S. aureus in food.