Guidelines for Light‐Start One‐Pot RPA‐CRISPR‐Cas12a Assays for Foodborne Pathogen Detection

ABSTRACT Rapid and accurate detection of nucleic acids of foodborne pathogens is of vital importance for controlling the spread of foodborne diseases and safeguarding public health. Recent advances in CRISPR based on recombinase polymerase amplification (RPA) have demonstrated the potential for highly specific and sensitivity molecular detection, but it faces obstacles such as distribution operations and contamination risks in practical applications. Light‐start CRISPR‐Cas12a reaction by CRISPR RNA (crRNA) conjugated with photocleaved modifications effectively resolves the incompatibility between the RPA system and the CRISPR system and has been gradually applied in the field of pathogen detection. Here, we provide operational guidelines for light‐start one‐pot RPA‐CRISPR‐Cas12a systems for foodborne pathogen detection. In this guideline, taking Listeria monocytogenes as an example, we provide experimental details on the design of photocleaved modified crRNA. Subsequently, we focus on elucidating the mechanisms of two light‐start one‐pot RPA‐CRISPR‐Cas12a assays based on different designs of crRNA, with comparative analysis against conventional two‐step and conventional one‐pot assay. Finally, these assays are thoroughly evaluated for analytical performance and practical applicability. This guideline aims to address operational challenges in biosensor development and point‐of‐care testing (POCT) applications, serving as a practical technical reference for researchers conducting foodborne pathogen detection experiments.