Related Experiment Video
Updated: Mar 31, 2026
Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System
Published on: April 25, 2025
Development of a rapid recombinase polymerase amplification assay for the detection of Streptococcus pneumoniae in
Eoin Clancy1,2, Owen Higgins3,4, Matthew S Forrest5
1Molecular Diagnostics Research Group, School of Natural Sciences, National University of Ireland, Galway, Ireland. eoin.clancy@nuigalway.ie.
Background:
Streptococcus pneumoniae is an important cause of microbial disease in humans. The introduction of multivalent vaccines has coincided with a dramatic decrease in the number of pneumococcal-related deaths. In spite of this, at a global level, pneumococcal infection remains an important cause of death among children under 5 years of age and in adults 65 years of age or older. In order to properly manage patients and control the spread of infection, a rapid and highly sensitive diagnostic method is needed for routine implementation, especially in resource-limited regions where pneumococcal disease is most prevalent.
Methods:
Using the gene encoding leader peptidase A as a molecular diagnostics target, a real-time RPA assay was designed and optimised for the detection of S. pneumoniae in whole blood. The performance of the assay was compared to real-time PCR in terms of its analytical limit of detection and specificity. The inhibitory effect of human genomic DNA on amplification was investigated. The potential clinical utility of the assay was investigated using a small number of clinical samples.
Results:
The RPA assay has a limit of detection equivalent to PCR (4.0 and 5.1 genome equivalents per reaction, respectively) and was capable of detecting the equivalent of <1 colony forming unit of S. pneumoniae when spiked into human whole blood. The RPA assay was 100 % inclusive (38/38 laboratory reference strains and 19/19 invasive clinical isolates) and 100 % exclusive; differentiating strains of S. pneumoniae species from other viridans group streptococci, including S. pseudopneumoniae. When applied to the analysis of a small number (n = 11) of clinical samples (blood culture positive for S. pneumoniae), the RPA assay was demonstrated to be both rapid and sensitive.
Conclusions:
The RPA assay developed in this work is shown to be as sensitive and as specific as PCR. In terms of reaction kinetics, the RPA assay is shown to exceed those of the PCR, with the RPA running to completion in 20 minutes and capable generating a positive signal in as little as 6 minutes. This work represents a potentially suitable assay for application in point-of-care settings.
Insights
A new real-time RPA assay can rapidly and sensitively detect Streptococcus pneumoniae in whole blood, matching PCR's accuracy. This diagnostic tool shows promise for point-of-care settings, especially in resource-limited areas.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Disease Research
Background:
- Streptococcus pneumoniae causes significant global mortality, particularly in young children and the elderly.
- Despite vaccines, pneumococcal disease remains a major public health concern.
- Rapid and sensitive diagnostics are crucial for managing infections, especially in resource-limited settings.
Purpose of the Study:
- To develop and optimize a real-time RPA assay for detecting S. pneumoniae in whole blood.
- To compare the RPA assay's performance against real-time PCR.
- To evaluate the assay's potential for clinical application.
Main Methods:
- Real-time RPA assay designed targeting the leader peptidase A gene.
- Analytical limit of detection and specificity compared to real-time PCR.
- Investigation of human genomic DNA's inhibitory effect on amplification.
- Assessment of clinical utility using a small cohort of patient samples.
Main Results:
- RPA assay demonstrated a limit of detection comparable to PCR (4.0-5.1 genome equivalents/reaction).
- The assay detected less than 1 CFU of S. pneumoniae in spiked whole blood.
- 100% inclusivity and exclusivity were observed, differentiating S. pneumoniae from other streptococci.
- RPA assay proved rapid and sensitive in preliminary clinical sample analysis.
Conclusions:
- The developed RPA assay is as sensitive and specific as PCR for S. pneumoniae detection.
- RPA assay exhibits faster kinetics than PCR, with results in as little as 6 minutes and completion in 20 minutes.
- This RPA assay is a potentially suitable diagnostic tool for point-of-care applications.
