One-Tube RPA-CRISPR/Cas12a Assays for Rapid and Visual Detection of Pseudomonas fluorescens and Bacillus cereus

Bacilus cereus and Pseudomonas fluorescens are major foodborne psychrotrophic bacteria posing global health and economic risks. B. cereus has a 23.8% food prevalence worldwide. P. fluorescens is a leading cause of spoilage in refrigerated products. Their rapid detection is crucial for food safety. However, existing detection methods often rely on open-tube operations, risking aerosol contamination. In this study, we developed two independent one-tube RPA-CRISPR/Cas12a visual detection assays for B. cereus and P. fluorescens. Using a physical separation design, the recombinase polymerase amplification (RPA) and CRISPR/Cas12a detection were pre-assembled in a single reaction tube. After incubation, a brief centrifugation combined the components for enclosed detection. This step is compatible with portable mini-centrifuges. The assays can be completed within 40 min at 37 °C, with results visualized directly under blue light. Both assays demonstrated good specificity against six common non-target pathogens. The visual detection limits were 5.1 × 101 copies/μL for B. cereus and 2.1 × 101 copies/μL for P. fluorescens. Each assay was applied to 14 types of real-world food samples (naturally contaminated and uncontaminated, confirmed by PCR), achieving 100% concordance with conventional PCR. The one-tube assays are tailored for psychrotrophic bacteria in refrigerated foods. They minimize aerosol contamination risk and provide a reliable solution for on-site cold-chain food safety monitoring.