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Rapid, sensitive, and user-friendly detection of Pseudomonas aeruginosa using the RPA/CRISPR/Cas12a system
Wenjing Zhang1, Hai Qu2, Xin Wu3
1Medical college, Henan University of Chinese Medicine, No.156, Jinshui East Road, Zhengzhou, 450046, Henan, China. hnzysophia@163.com.
Background:
Pseudomonas aeruginosa (P. aeruginosa) is a life-threatening bacterium known for its rapid development of antibiotic resistance, posing significant challenges in clinical treatment, biosecurity, food safety, and environmental monitoring. Early and accurate identification of P. aeruginosa is crucial for effective intervention.
Methods:
The lasB gene of P. aeruginosa was selected as the target for the detection. RPA primers for recombinase polymerase amplification (RPA) and crRNA for CRISPR/Cas12a detection were meticulously designed to target specific regions within the lasB gene. The specificity of the RPA/CRISPR/Cas12a detection platform was assessed using 15 strains. The detection limit of RPA/CRISPR/Cas12a detection platform was determined by utilizing a pseudo-dilution series of the P. aeruginosa DNA. The practical applicability of the RPA/CRISPR/Cas12a detection platform was validated by comparing it with qPCR on 150 samples (35 processed meat product samples, 55 cold seasoned vegetable dishes, 60 bottled water samples).
Results:
The RPA/CRISPR/Cas12a detection platform demonstrates high specificity, with no cross-reactivity with non-P. aeruginosa strains. This assay exhibits remarkable sensitivity, with a limit of detection (LOD) of 100 copies/µL for fluorescence assay and 101 copies/µL for the LFTS method. Furthermore, the performance of the RPA/CRISPR/Cas12a detection platform is comparable to that of the well-established qPCR method, while offering advantages such as shorter reaction time, simplified operation, and reduced equipment requirements.
Conclusions:
The RPA/CRISPR/Cas12a detection platform presents a straightforward, accurate, and sensitive approach for early P. aeruginosa detection and holds great promise for diverse applications requiring rapid and reliable identification.
Insights
A new RPA/CRISPR/Cas12a detection platform offers rapid and accurate identification of Pseudomonas aeruginosa. This sensitive method is crucial for clinical, food, and environmental safety applications.
Area of Science:
- Microbiology
- Molecular Biology
- Biotechnology
Background:
- Pseudomonas aeruginosa (P. aeruginosa) is a dangerous bacterium known for rapid antibiotic resistance.
- This resistance poses challenges in clinical settings, biosecurity, food safety, and environmental monitoring.
- Early and accurate P. aeruginosa detection is vital for effective interventions.
Purpose of the Study:
- To develop and validate a novel detection platform for P. aeruginosa.
- To target the lasB gene for sensitive and specific identification.
- To assess the platform's performance against established methods like qPCR.
Main Methods:
- Designed RPA primers and CRISPR/Cas12a crRNA targeting the P. aeruginosa lasB gene.
- Evaluated specificity using 15 bacterial strains and determined the limit of detection (LOD) via DNA dilution series.
- Validated practical applicability by comparing the platform with qPCR on 150 diverse samples.
Main Results:
- The RPA/CRISPR/Cas12a platform showed high specificity with no cross-reactivity.
- Achieved a low LOD of 10^0 copies/µL (fluorescence) and 10^1 copies/µL (LFTS).
- Demonstrated comparable performance to qPCR, with advantages in speed, simplicity, and equipment needs.
Conclusions:
- The RPA/CRISPR/Cas12a platform provides a simple, accurate, and sensitive method for early P. aeruginosa detection.
- This approach is promising for various applications needing rapid and reliable bacterial identification.
- The platform facilitates timely interventions in clinical, food safety, and environmental contexts.
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