Development of internally controlled duplex real-time NASBA diagnostics assays for the detection of microorganisms associated with bacterial meningitis.
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.
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.
Eoin
E
Clancy
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.
Helena
H
Coughlan
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.
Owen
O
Higgins
Department of Clinical Microbiology, University College Hospital, Galway, Ireland.
Teck Wee
TW
Boo
School of Medicine, National University of Ireland, Galway, Ireland.
Martin
M
Cormican
National Centre of Sensor Research, School of Physical Sciences, Biomedical Diagnostics Institute, Dublin City University, Glasnevin, Dublin 9, Ireland.
Louise
L
Barrett
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.
Terry J
TJ
Smith
Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
Kate
K
Reddington
Nucleic Acid Diagnostics Research Laboratory (NADRL), Microbiology, School of Natural Sciences, National University of Ireland, Galway, Ireland.
Thomas
T
Barry
D006193:Haemophilus influenzae / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D057230:Limit of Detection; D016920:Meningitis, Bacterial / Q000175:diagnosis / Q000382:microbiology*; D008583:Meningitis, Haemophilus / Q000175:diagnosis / Q000382:microbiology; D008585:Meningitis, Meningococcal / Q000175:diagnosis / Q000382:microbiology; D008586:Meningitis, Pneumococcal / Q000175:diagnosis / Q000382:microbiology; D009345:Neisseria meningitidis / Q000235:genetics / Q000302:isolation & purification*; D021161:Self-Sustained Sequence Replication / Q000379:methods*; D012680:Sensitivity and Specificity; D013296:Streptococcus pneumoniae / Q000235:genetics / Q000302:isolation & purification*; D035181:TATA-Box Binding Protein / Q000235:genetics
D035181:TATA-Box Binding Protein; C509817:TBP protein, human
Bacterial meningitis; Haemophilus influenzae; IAC; Neisseria meningitidis; Real-time NASBA; Streptococcus pneumoniae