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Development and performance evaluation of a recombinase polymerase amplification assay for the rapid detection of
Christina Clarke1, Louise O'Connor2, Heather Carré-Skinner3
1Molecular Diagnostics Research Group, School of Natural Sciences, National Centre for Biomedical Engineering Science (NCBES), National University of Ireland, Galway, Ireland.
Insights
A new Recombinase Polymerase Amplification (RPA) assay accurately detects group B Streptococcus (GBS) in vaginal swabs. This sensitive and specific test offers comparable performance to PCR, aiding in timely GBS diagnosis during delivery.
Area of Science:
- Microbiology
- Molecular Diagnostics
- Infectious Diseases
Background:
- Group B Streptococcus (GBS) causes significant neonatal sepsis, pneumonia, and meningitis.
- Current GBS prevention guidelines have reduced but not eliminated neonatal GBS disease.
- A rapid, near-patient diagnostic test for GBS at delivery is needed, especially for unknown colonization status.
Purpose of the Study:
- To develop and evaluate a Recombinase Polymerase Amplification (RPA) assay for detecting GBS in vaginal swabs.
- To assess the analytical and clinical performance of the novel RPA assay.
Main Methods:
- Developed an RPA assay targeting the cAMP factor (cfb) gene of GBS.
- Evaluated analytical specificity using GBS reference strains, clinical isolates, and non-GBS organisms.
- Determined the limit of detection and assessed clinical performance with 124 vaginal swabs.
Main Results:
- The RPA assay demonstrated high specificity for GBS.
- The limit of detection was 6-12 genome copies, comparable to real-time PCR (<12.5 genome copies).
- Clinical performance of the RPA assay was identical to real-time PCR.
Conclusions:
- A specific and sensitive RPA assay for GBS detection was successfully developed.
- The assay's performance in clinical samples is acceptable for near-patient GBS diagnostics.
Background:
Despite the implementation of prevention guidelines, group B Streptococcal (GBS) infection remains a leading cause of sepsis, pneumonia, and meningitis, resulting in significant neonatal morbidity and mortality. Preventive approaches that identify women at risk of transmitting GBS have reduced the incidence of neonatal GBS disease, and dramatically decreased the associated mortality rates. However, there is an on-going requirement for a near-patient diagnostic test for GBS that can be carried out at the time of delivery, ideally in the labour ward setting, particularly for women of unknown GBS colonisation status at the time of delivery.
Methods:
In this study, a Recombinase Polymerase Amplification (RPA) assay was developed and performance evaluated for the detection of group B Streptococcus in vaginal swabs. The assay uses the cAMP factor (cfb) gene of GBS as the target gene. The analytical performance of the assay was evaluated by testing a panel of GBS reference strains and clinical isolates, and non-GBS organisms. The limit of detection was determined and the clinical performance was evaluated by testing 124 vaginal swabs from women with both GBS positive and negative status.
Results:
Based on specificity testing carried out the assay was shown to be specific for the target of interest. The limit of detection of the assay was shown to be between six and 12 genome copies and was comparable to that of a real-time PCR assay, both achieving a limit of detection below 12.5 genome copies. The performance of both assays when applied to clinical samples was identical.
Conclusion:
A specific, sensitive RPA assay for GBS was developed. The performance of the assay for testing of clinical samples is within the acceptable range.
