Method development and clinical validation of LAMP-CRISPR/Cas12a for rapid detection of respiratory pathogens in
Siyan Zhou1, Xin Zhao1, Fanzheng Meng1,2
1Pediatric Department of Respiration, The First Bethune Hospital of Jilin University, Changchun, China.
Insights
A new diagnostic method using loop-mediated isothermal amplification (LAMP) and CRISPR-Cas12a rapidly and accurately detects Streptococcus pneumoniae (SP) and Mycoplasma pneumoniae (MP) in respiratory infections. This advancement aids in timely treatment and improved patient outcomes.
Area of Science:
- Molecular diagnostics
- Infectious disease detection
- Biotechnology
Background:
- Respiratory tract infections (RTIs) present a significant global health challenge, especially in children.
- Accurate identification of pathogens like Streptococcus pneumoniae (SP) and Mycoplasma pneumoniae (MP) is vital for effective treatment.
- Current diagnostic methods may lack the speed or specificity required for timely clinical decisions.
Purpose of the Study:
- To develop and evaluate a novel diagnostic approach for detecting SP and MP in respiratory samples.
- To combine loop-mediated isothermal amplification (LAMP) with CRISPR-Cas12a technology for enhanced pathogen detection.
- To assess the feasibility and performance of this integrated molecular diagnostic method.
Main Methods:
- A novel diagnostic assay integrating LAMP amplification with CRISPR-Cas12a detection was developed.
- Nucleic acids were extracted from 23 clinical respiratory samples (bronchoalveolar lavage, nasopharyngeal swabs).
- Samples underwent LAMP amplification followed by CRISPR-Cas12a-mediated fluorescence detection for SP and MP identification.
Main Results:
- The LAMP-CRISPR/Cas12a method showed high sensitivity and specificity for detecting Streptococcus pneumoniae.
- Promising sensitivity and specificity were observed for Mycoplasma pneumoniae detection.
- The assay provided results within one hour, indicating suitability for rapid point-of-care testing.
Conclusions:
- The integrated LAMP-CRISPR/Cas12a approach offers a rapid, sensitive, and specific tool for diagnosing respiratory pathogens.
- This molecular diagnostic strategy facilitates early and precise identification of SP and MP, crucial for guiding precision therapies.
- Future work will focus on optimizing sensitivity, minimizing contamination, and expanding pathogen detection capabilities.
Background:
Respiratory tract infections pose a substantial health burden, particularly among pediatric populations globally. The timely and accurate identification of pathogens such as Streptococcus pneumoniae (SP) and Mycoplasma pneumoniae (MP) is critical for effective clinical management.
Methods:
In this study, a novel diagnostic approach combining loop-mediated isothermal amplification (LAMP) with CRISPR-Cas12a technology was developed for detecting SP and MP in clinical respiratory samples. A total of 23 specimens, including bronchoalveolar lavage fluid and nasopharyngeal swab samples, were assessed to evaluate the feasibility and performance of the method. After nucleic acid extraction, samples underwent LAMP amplification followed by CRISPR-Cas12a-mediated fluorescence detection.
Results:
The LAMP-CRISPR/Cas12a method demonstrated high sensitivity and specificity for SP detection. It exhibited excellent sensitivity for SP and promising specificity for MP. Comparative analysis with standard diagnostic methods highlighted its potential to enhance diagnostic accuracy and efficiency. The assay provided results within 1 h, which is suitable for rapid point-of-care testing.
Conclusion:
The integrated LAMP-CRISPR/Cas12a approach represents a significant advancement in detecting respiratory pathogens in clinical settings. It offers a rapid, sensitive, and specific diagnostic tool for identifying SP and MP, which is crucial for guiding precision therapies and improving patient outcomes. Future research aims to optimize assay sensitivity, streamline workflow to minimize contamination risks, and expand its detection scope so that other types of pathogens and mutation resistance genes can be detected. This molecular diagnostic strategy holds promise for the management of respiratory infections by enabling early and precise pathogen identification.
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