A one-pot method based on rolling circle amplification and light‐activated CRISPR/Cas12a reaction for simple and highly sensitive detection of Staphylococcus aureus

Developing robust methods that achieve simple and highly sensitive detection of Staphylococcus aureus (S. aureus) is still a challenge. Isothermal nucleic acid amplification reactions-triggered CRISPR/Cas12a strategies offer promising sensing tools for bacteria detection, but have several issues that need to be solved, such as incompatible reaction systems, compromised sensitivity, and multiple reaction steps. Here, we develop a light-activated one-pot method based on rolling circle amplification-triggered CRISPR/Cas12a (RCA-Cas12a) for analyzing S. aureus directly. In the one-pot assay, the Cas12a reaction is started under light irradiation only after the RCA reaction is completed, thus avoiding interference between the two systems. The light-activated method is simple to operate, and its sensitivity is two orders of magnitude higher than that of the traditional one-pot assay. Accurately identifying contaminated samples from 120 real samples (serum, milk, and broth) prepared in a double-blind way demonstrates the good application capacity of the light-activated one-pot method. The method has a shorter analysis time, fewer operation steps, and higher accuracy in identifying contaminated samples compared with bacterial culture. Thus, the light-activated one-pot RCA-Cas12a method represents a simple tool for sensitive and specific detection of S. aureus in food and clinical samples.