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Duplex recombinase polymerase amplification assays incorporating competitive internal controls for bacterial meningitis detection.

Authors
  • Higgins, Owen1
  • Clancy, Eoin2
  • Forrest, Matthew S3
  • Piepenburg, Olaf3
  • Cormican, Martin4
  • Boo, Teck Wee4
  • O'Sullivan, Nicola5
  • McGuinness, Claire5
  • Cafferty, Deirdre5
  • Cunney, Robert5
  • Smith, Terry J2
  • 1 Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland. Electronic address: [email protected] , (Ireland)
  • 2 Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland. , (Ireland)
  • 3 TwistDx Limited, Cambridge, United Kingdom. , (United Kingdom)
  • 4 School of Medicine, Galway University Hospital, National University of Ireland, Galway, Ireland. , (Ireland)
  • 5 Irish Meningitis and Sepsis Reference Laboratory, Temple Street Children's University Hospital, Temple Street, Dublin, Ireland. , (Ireland)
Type
Published Article
Journal
Analytical Biochemistry
Publisher
Elsevier
Publication Date
Jan 30, 2018
Volume
546
Pages
10–16
Identifiers
DOI: 10.1016/j.ab.2018.01.016
PMID: 29378166
Source
Medline
Keywords
License
Unknown

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.

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