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

## TL;DR

This paper introduces a fast and safe method to detect two harmful bacteria in refrigerated foods using a single-tube CRISPR-based assay.

## Contribution

The novel one-tube RPA-CRISPR/Cas12a assays reduce contamination risk and enable rapid on-site detection of B. cereus and P. fluorescens.

## Key findings

- The assays achieved 100% agreement with PCR in 14 real-world food samples.
- Detection limits were 5.1 × 10¹ copies/μL for B. cereus and 2.1 × 10¹ copies/μL for P. fluorescens.
- The method is compatible with portable mini-centrifuges and provides results in 40 minutes at 37°C.

## Abstract

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.