Article ID | Journal | Published Year | Pages | File Type |
---|---|---|---|---|
7556985 | Analytical Biochemistry | 2018 | 25 Pages |
Abstract
Recombinase polymerase amplification (RPA) is an isothermal nucleic acid amplification technology that provides rapid and robust infectious disease pathogen detection, ideal for point-of-care (POC) diagnostics in disease-prevalent low-resource countries. We have developed and evaluated three duplex RPA assays incorporating competitive internal controls for the detection of leading bacterial meningitis pathogens. Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae singleplex RPA assays were initially developed and evaluated, demonstrating 100% specificity with limits of detection of 4.1, 8.5 and 3.9 genome copies per reaction, respectively. Each assay was further developed into internally controlled duplex RPA assays via the incorporation of internal amplification control templates. Clinical performance of each internally controlled duplex RPA assay was evaluated by testing 64 archived PCR-positive clinical samples. Compared to real-time PCR, all duplex RPA assays demonstrated 100% diagnostic specificity, with diagnostic sensitivities of 100%, 86.3% and 100% for the S. pneumoniae, N. meningitidis and H. influenzae assays, respectively. This study details the first report of internally controlled duplex RPA assays for the detection of bacterial meningitis pathogens: S. pneumoniae, N. meningitidis and H. influenzae. We have successfully demonstrated the clinical diagnostic utility of each duplex RPA assay, introducing effective diagnostic technology for POC bacterial meningitis identification in disease-prevalent developing countries.
Keywords
RFUBHIRPAHDAIACPOCTHFNTCDNAdeoxyribonucleic acidNo template controlTetrahydrofuranbrain heart infusionLoop-mediated isothermal amplificationRecombinase polymerase amplificationLOD یا Limit of detectionDuplexLAMPCSFCerebrospinal fluidlimit of detectionBacterial meningitispoint-of-careRelative Fluorescence Unitspolymerase chain reactionPCRPirBhigh performance liquid chromatographyHPLCInternal amplification control
Related Topics
Physical Sciences and Engineering
Chemistry
Analytical Chemistry
Authors
Owen Higgins, Eoin Clancy, Matthew S. Forrest, Olaf Piepenburg, Martin Cormican, Teck Wee Boo, Nicola O'Sullivan, Claire McGuinness, Deirdre Cafferty, Robert Cunney, Terry J. Smith,