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Rapid detection of Mycobacterium tuberculosis using recombinase polymerase amplification: A pilot study
Michael Sciaudone1,2, Renzo Carpena3, Maritza Calderón3
1Department of Medicine, Section of Infectious Diseases, Tulane University School of Medicine, New Orleans, Louisiana, United States of America.
Plos One
|December 8, 2023
Summary
Recombinase polymerase amplification (RPA) offers a rapid, cost-effective method for diagnosing tuberculosis. The real-time RPA assay shows promise for resource-limited settings, while the lateral flow assay requires further optimization.
Area of Science:
- Molecular Biology
- Infectious Disease Diagnostics
- Biotechnology
Background:
- Tuberculosis (TB) is a major global health threat, particularly in low- and middle-income countries.
- Current TB diagnostic methods are often slow, expensive, or lack sufficient sensitivity, hindering effective control.
- Recombinase polymerase amplification (RPA) is a promising isothermal DNA amplification technique for rapid diagnostics.
Purpose of the Study:
- To evaluate and compare the diagnostic performance of two RPA-based assays for pulmonary tuberculosis.
- To assess the limit of detection, sensitivity, and specificity of RPA-lateral flow and real-time RPA assays.
- To compare the impact of two DNA extraction methods (commercial kit vs. Chelex) on RPA assay performance.
Main Methods:
- Two sets of published primers were used to develop RPA assays for pulmonary tuberculosis detection.
- Assays were tested using stored sputum specimens, comparing a lateral flow format with a real-time format.
- DNA was extracted using a commercial kit and the Chelex method to evaluate their influence on assay results.
Main Results:
- The RPA-lateral flow assay exhibited a limit of detection of 5 fg/μL, 53.2% sensitivity, and 93.3% specificity.
- The real-time RPA assay demonstrated a limit of detection of 25 fg/μL, 85.1% sensitivity, and 93.3% specificity.
- No significant difference in assay performance was observed between the commercial DNA extraction kit and the Chelex method.
Conclusions:
- The real-time RPA assay demonstrates sufficient sensitivity and specificity for pulmonary tuberculosis diagnosis, presenting a viable option for resource-limited settings.
- The RPA-lateral flow assay's performance was suboptimal, potentially due to the use of stored specimens; further optimization is required.
- Both RPA assays require further development, including validation with prospective specimens and adaptation into point-of-care tests for field deployment.
