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Updated: Feb 11, 2026
Development of a Quantitative Recombinase Polymerase Amplification Assay with an Internal Positive Control
Published on: March 30, 2015
A recombinase polymerase amplification assay for the diagnosis of atypical pneumonia
Sebastian Kersting1, Valentina Rausch1, Frank F Bier2
1Fraunhofer Institute for Cell Therapy and Immunology, Branch Bioanalytics and Bioprocesses (Fraunhofer IZI-BB), Germany.
Abstract:
Pneumonia is one of the most common and potentially lethal infectious conditions worldwide. Streptococcus pneumoniae is the pathogen most frequently associated with bacterial community-acquired pneumonia, while Legionella pneumophila is the major cause for local outbreaks of legionellosis. Both pathogens can be difficult to diagnose since signs and symptoms are nonspecific and do not differ from other causes of pneumonia. Therefore, a rapid diagnosis within a clinically relevant time is essential for a fast onset of the proper treatment. Although methods based on polymerase chain reaction significantly improved the identification of pathogens, they are difficult to conduct and need specialized equipment. We describe a rapid and sensitive test using isothermal recombinase polymerase amplification and detection on a disposable test strip. This method does not require any special instrumentation and can be performed in less than 20 min. The analytical sensitivity in the multiplex assay amplifying specific regions of S. pneumoniae and L. pneumophila simultaneously was 10 CFUs of genomic DNA per reaction. In cross detection studies with closely related strains and other bacterial agents the specificity of the RPA was confirmed. The presented method is applicable for near patient and field testing with a rather simple routine and the possibility for a read out with the naked eye.
Insights
A new rapid diagnostic test accurately detects Streptococcus pneumoniae and Legionella pneumophila, the main causes of pneumonia. This simple, instrument-free method provides results in under 20 minutes for faster treatment.
Area of Science:
- Microbiology
- Infectious Diseases
- Biotechnology
Background:
- Pneumonia is a leading cause of infectious death globally.
- Streptococcus pneumoniae and Legionella pneumophila are key bacterial pathogens responsible for pneumonia and legionellosis outbreaks.
- Current diagnostic methods for pneumonia lack speed and require specialized equipment, delaying treatment.
Purpose of the Study:
- To develop a rapid, sensitive, and specific diagnostic test for S. pneumoniae and L. pneumophila.
- To create a method suitable for near-patient and field testing without specialized instrumentation.
- To enable timely diagnosis and treatment initiation for pneumonia patients.
Main Methods:
- Utilized isothermal recombinase polymerase amplification (RPA) for simultaneous detection of S. pneumoniae and L. pneumophila.
- Developed a multiplex assay targeting specific genomic regions of both pathogens.
- Employed a disposable test strip for easy detection, readable by the naked eye.
Main Results:
- Achieved analytical sensitivity of 10 CFUs of genomic DNA per reaction for the multiplex RPA assay.
- Confirmed high specificity through cross-detection studies with related strains and other bacteria.
- Demonstrated the method's applicability for rapid, instrument-free testing in under 20 minutes.
Conclusions:
- The developed RPA test offers a rapid, sensitive, and specific tool for diagnosing S. pneumoniae and L. pneumophila infections.
- This technology is suitable for point-of-care and field diagnostics, overcoming limitations of existing methods.
- The test's simplicity and speed facilitate prompt clinical decision-making for pneumonia management.
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