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Development of a Rapid and Efficient RPA-CRISPR/Cas12a Assay for Mycoplasma pneumoniae Detection
Feina Li1, Jing Xiao1, Haiming Yang2
1Laboratory of Respiratory Diseases, Beijing Key Laboratory of Pediatric Respiratory Infection Diseases, Beijing Pediatric Research Institute, Beijing Children's Hospital, Capital Medical University, Key Laboratory of Major Diseases in Children, Ministry of Education, National Clinical Research Center for Respiratory Diseases, National Center for Children's Health, Beijing, China.
Abstract:
Mycoplasma pneumoniae (MP) is a one of most common pathogen in causing respiratory infection in children and adolescents. Rapid and efficient diagnostic methods are crucial for control and treatment of MP infections. Herein, we present an operationally simple, rapid and efficient molecular method for MP identification, which eliminates expensive instruments and specialized personnel. The method combines recombinase polymerase amplification (RPA) with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated proteins (Cas) 12a-based detection, with an optimal procedure less than 1 h from sample to result including DNA extraction (25 min), RPA reaction (39°C for 15-20 min), CRISPR/Cas12a detection (37°C for 10 min) and visual detection by naked eyes (2 min). This diagnostic method shows high sensitivity (two copies per reaction) and no cross-reactivity against other common pathogenic bacteria. Preliminary evaluation using 201 clinical samples shows sensitivity of 99.1% (107/108), specificity of 100% (93/93) and consistency of 99.5% (200/201), compared with real-time PCR method. The above data demonstrate that our developed method is reliable for rapid diagnosis of MP. In conclusion, the RPA-CRISPR/Cas12a has a great potential to be as a useful tool for reliable and quick diagnosis of MP infection, especially in primary hospitals with limited conditions.
Insights
A new molecular diagnostic method combines recombinase polymerase amplification (RPA) and CRISPR/Cas12a for rapid Mycoplasma pneumoniae (MP) detection. This efficient test provides accurate results in under an hour, ideal for primary healthcare settings.
Area of Science:
- Molecular Biology
- Infectious Diseases
- Diagnostics
Background:
- Mycoplasma pneumoniae (MP) is a common cause of respiratory infections in children and adolescents.
- Rapid and accurate diagnostic methods are essential for managing MP infections.
- Existing methods may require specialized equipment or personnel, limiting accessibility.
Purpose of the Study:
- To develop and evaluate a rapid, simple, and efficient molecular diagnostic method for MP identification.
- To utilize recombinase polymerase amplification (RPA) combined with CRISPR/Cas12a detection.
- To assess the method's performance in terms of speed, sensitivity, specificity, and cross-reactivity.
Main Methods:
- DNA extraction followed by RPA amplification at 39°C for 15-20 minutes.
- CRISPR/Cas12a-based detection at 37°C for 10 minutes.
- Visual readout by naked eye in 2 minutes, with a total assay time under 1 hour.
Main Results:
- The method demonstrated high sensitivity (2 copies/reaction) and no cross-reactivity with other common pathogens.
- Clinical evaluation on 201 samples showed 99.1% sensitivity and 100% specificity compared to real-time PCR.
- The overall consistency with real-time PCR was 99.5%.
Conclusions:
- The developed RPA-CRISPR/Cas12a method is a reliable tool for rapid MP diagnosis.
- Its operational simplicity and speed make it suitable for resource-limited settings, such as primary hospitals.
- This approach offers a promising alternative for quick and accurate identification of MP infections.
