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Klebsiella pneumoniae detection by a light-controlled one-pot RPA-CRISPR/Cas12a method
Lele Pan1,2,3,4,5, Lijian Wei1,2,3,4,5,6, Shihua Luo1,2,3,4,5
1Center for Medical Laboratory Science, Affiliated Hospital of Youjiang Medical University for Nationalities, Baise, Guangxi, China.
Background:
Klebsiella pneumoniae (KP) is a significant pathogenic bacterium responsible for severe infections in hospitals. However, existing traditional detection techniques, such as culture and PCR, are relatively inefficient. Therefore, this study aims to establish a rapid and convenient method for detecting KP.
Methods:
This study developed a single-tube detection method combining recombinant polymerase amplification (RPA) and light-controlled CRISPR/Cas12a. RPA primers were designed and screened for the rcsA gene of KP to effectively amplify the target. A light-controlled CRISPR/Cas12a system was created using crRNA modified with a photocleavable group (NPOM). The two systems were integrated into a single tube. Following RPA amplification, UV light-controlled release of crRNA inhibition activates CRISPR-mediated target recognition and Cas12a trans-cleavage, detecting fluorescent signals (FD) in conjunction with UV analysis.
Results:
The light-controlled RPA-CRISPR/Cas12a detection platform developed in this study uses a 15 μL reaction system. By optimizing key parameters such as RPA amplification time (20 min), primer concentration (400 nM), UV light activation time (30 s), and crRNA/Cas12a concentration (300 nM), the platform achieves optimal detection efficiency. The platform has a fluorescence detection limit of 4.072×102 copies/reaction and can specifically identify KP in seven common clinical strains. Clinical sample validation demonstrated that the method yields results fully consistent with PCR detection (30/30 agreement rate of 100%), showcasing excellent detection performance and clinical application potential.
Conclusion:
We have successfully developed a light-controlled RPA-CRISPR/Cas12a detection system capable of rapidly and highly sensitively detecting KP. This system demonstrates significant advantages in terms of detection speed (completed in as little as 50 minutes), sensitivity (as low as 4.072×102 copies/reaction), and ease of use, providing an efficient and reliable solution for clinical pathogen detection.
Insights
A new light-controlled RPA-CRISPR/Cas12a method rapidly detects Klebsiella pneumoniae (KP). This sensitive and user-friendly system offers a promising solution for clinical pathogen identification.
Area of Science:
- Molecular Biology
- Biotechnology
- Medical Diagnostics
Background:
- Klebsiella pneumoniae (KP) is a major cause of hospital-acquired infections.
- Traditional detection methods like culture and PCR are often slow and inefficient.
- There is a need for rapid and convenient KP detection techniques.
Purpose of the Study:
- To develop a novel, rapid, and sensitive detection method for Klebsiella pneumoniae.
- To integrate recombinant polymerase amplification (RPA) with a light-controlled CRISPR/Cas12a system.
- To establish a user-friendly platform for clinical pathogen detection.
Main Methods:
- A single-tube assay combining RPA and light-controlled CRISPR/Cas12a was developed.
- RPA primers targeted the rcsA gene of KP for amplification.
- A photocleavable group (NPOM) on crRNA enabled UV light-controlled CRISPR/Cas12a activation.
- The system was optimized for reaction time, primer concentration, and UV activation parameters.
Main Results:
- The optimized platform achieved a detection limit of 4.072×10^2 copies/reaction.
- The method demonstrated high specificity, identifying KP in seven common clinical strains.
- Clinical sample validation showed 100% agreement with PCR results (30/30).
- The entire detection process was completed in as little as 50 minutes.
Conclusions:
- A light-controlled RPA-CRISPR/Cas12a system for rapid and sensitive KP detection was successfully established.
- The system offers significant advantages in speed, sensitivity, and ease of use.
- This technology presents a reliable and efficient solution for clinical pathogen detection.
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