Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis

Eoin Clancy; Helena Coughlan; Owen Higgins; Teck Wee Boo; Martin Cormican; Louise Barrett; Terry J Smith; Kate Reddington; Thomas Barry

doi:10.1016/j.mimet.2016.06.017

Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis

J Microbiol Methods. 2016 Aug:127:197-202. doi: 10.1016/j.mimet.2016.06.017. Epub 2016 Jun 16.

Authors

Eoin Clancy¹, Helena Coughlan², Owen Higgins¹, Teck Wee Boo³, Martin Cormican⁴, Louise Barrett⁵, Terry J Smith¹, Kate Reddington⁶, Thomas Barry⁶

Affiliations

¹ Biomedical Diagnostics Institute Programme, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland; Molecular Diagnostics Research Group (MDRG), School of Natural Sciences, National University of Ireland, Galway, Ireland.
² Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland; Biomedical Diagnostics Institute Programme, National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland.
³ Department of Clinical Microbiology, University College Hospital, Galway, Ireland.
⁴ School of Medicine, National University of Ireland, Galway, Ireland.
⁵ National Centre of Sensor Research, School of Physical Sciences, Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin 9, Ireland.
⁶ Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.

PMID: 27319375
DOI: 10.1016/j.mimet.2016.06.017

Abstract

Three duplex molecular beacon based real-time Nucleic Acid Sequence Based Amplification (NASBA) assays have been designed and experimentally validated targeting RNA transcripts for the detection and identification of Haemophilus influenzae, Neisseria meningitidis and Streptococcus pneumoniae respectively. Each real-time NASBA diagnostics assay includes an endogenous non-competitive Internal Amplification Control (IAC) to amplify the splice variant 1 mRNA of the Homo sapiens TBP gene from human total RNA. All three duplex real-time NASBA diagnostics assays were determined to be 100% specific for the target species tested for. Also the Limits of Detection (LODs) for the H. influenzae, N. meningitidis and S. pneumoniae duplex real-time NASBA assays were 55.36, 0.99, and 57.24 Cell Equivalents (CE) respectively. These robust duplex real-time NASBA diagnostics assays have the potential to be used in a clinical setting for the rapid (<60min) specific detection and identification of the most prominent microorganisms associated with bacterial meningitis in humans.

Keywords: Bacterial meningitis; Haemophilus influenzae; IAC; Neisseria meningitidis; Real-time NASBA; Streptococcus pneumoniae.

MeSH terms

Haemophilus influenzae / genetics
Haemophilus influenzae / isolation & purification*
Humans
Limit of Detection
Meningitis, Bacterial / diagnosis
Meningitis, Bacterial / microbiology*
Meningitis, Haemophilus / diagnosis
Meningitis, Haemophilus / microbiology
Meningitis, Meningococcal / diagnosis
Meningitis, Meningococcal / microbiology
Meningitis, Pneumococcal / diagnosis
Meningitis, Pneumococcal / microbiology
Neisseria meningitidis / genetics
Neisseria meningitidis / isolation & purification*
Self-Sustained Sequence Replication / methods*
Sensitivity and Specificity
Streptococcus pneumoniae / genetics
Streptococcus pneumoniae / isolation & purification*
TATA-Box Binding Protein / genetics

Substances

TATA-Box Binding Protein
TBP protein, human