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A phage amplification-assisted SEA-CRISPR/Cas12a system for viable bacteria detection
Xiangyang Xiao1, Chenlu Zhang1, Li Zhang1
1Key Laboratory of Medical Diagnostics of Ministry of Education, College of Laboratory Medicine, Chongqing Medical Laboratory Microfluidics and SPRi Engineering Research Center, Chongqing Medical University, Chongqing, 400016, P. R. China. yangyujun@cqmu.edu.cn.
This study presents a new phage-assisted isothermal platform using strand exchange amplification (SEA) and CRISPR/Cas12a for rapid, accurate detection of viable bacteria in urine. The method achieves a 10^3 CFU/mL detection limit in 3.5 hours without DNA extraction.
Area of Science:
- Molecular diagnostics
- Microbiology
- Biotechnology
Background:
- Accurate detection of viable bacteria is crucial for diagnosing urinary tract infections (UTIs) and guiding treatment.
- Current molecular methods often fail to distinguish between live and dead bacteria, impacting diagnostic accuracy.
Purpose of the Study:
- To develop a novel, rapid, and sensitive diagnostic platform for detecting viable bacteria in clinical samples.
- To overcome the limitations of existing methods in differentiating viable from non-viable bacterial cells.
Main Methods:
- Integration of strand exchange amplification (SEA) with the CRISPR/Cas12a system on an isothermal platform.
- Incorporation of bacteriophages for specific recognition and signal amplification of viable bacteria.
- Direct detection in human urine samples without prior DNA extraction.
Main Results:
- Achieved high sensitivity for phage DNA detection down to 4 × 10^2 copies/μL.
- Successfully detected viable bacteria in urine samples with a limit of 10^3 CFU/mL within 3.5 hours.
- Demonstrated specificity and amplification through phage properties.
Conclusions:
- The phage-assisted SEA-Cas12a platform offers rapid, accurate, and sensitive detection of viable bacteria.
- This method shows significant potential for point-of-care diagnostics, especially in resource-limited settings.
- Facilitates timely clinical management of UTIs and other bacterial infections.
