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One-Tube RPA-CRISPR/Cas12a Assays for Rapid and Visual Detection of Pseudomonas fluorescens and Bacillus cereus
Changli Yang1,2,3, Gaoke Wang1,2,3, Xiaowu Zhou2,3
1School of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China.
Insights
New one-tube assays rapidly detect foodborne bacteria Bacillus cereus and Pseudomonas fluorescens. These enclosed tests minimize contamination risk for reliable cold-chain food safety monitoring.
Area of Science:
- Food Science
- Microbiology
- Biotechnology
Background:
- Bacillus cereus and Pseudomonas fluorescens are prevalent psychrotrophic bacteria causing foodborne illnesses and spoilage.
- Current detection methods pose risks of aerosol contamination due to open-tube operations.
- Rapid and safe detection is essential for effective food safety management.
Purpose of the Study:
- To develop novel, one-tube visual detection assays for B. cereus and P. fluorescens.
- To enhance food safety by minimizing contamination risks during bacterial detection.
- To provide a rapid and reliable method for on-site cold-chain food monitoring.
Main Methods:
- Development of two independent Recombinase Polymerase Amplification (RPA)-CRISPR/Cas12a assays in a single tube.
- Utilized a physical separation design with pre-assembly and brief centrifugation for enclosed detection.
- Assays performed at 37 °C for 40 minutes with visual results under blue light.
Main Results:
- High specificity demonstrated against common non-target pathogens.
- Low visual detection limits achieved: 5.1 × 10^1 copies/μL for B. cereus and 2.1 × 10^1 copies/μL for P. fluorescens.
- 100% concordance with conventional PCR in 14 real-world food samples.
Conclusions:
- The developed one-tube RPA-CRISPR/Cas12a assays offer a safe and rapid method for detecting B. cereus and P. fluorescens.
- These assays are suitable for on-site monitoring of psychrotrophic bacteria in refrigerated foods.
- The enclosed system effectively minimizes aerosol contamination, enhancing laboratory and field safety.
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.
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