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

<i>Bacilus cereus</i> and <i>Pseudomonas fluorescens</i> are major foodborne psychrotrophic bacteria posing global health and economic risks. <i>B. cereus</i> has a 23.8% food prevalence worldwide. <i>P. fluorescens</i> 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 <i>B. cereus</i> and <i>P. fluorescens</i>. 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 <i>B. cereus</i> and 2.1 × 10¹ copies/μL for <i>P. fluorescens.</i> 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.