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Int. J. Mol. Sci. 2018, 19(2), 524; https://doi.org/10.3390/ijms19020524

Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens

1
Molecular Diagnostics Research Group, School of Natural Sciences and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Ireland
2
School of Medicine, National University of Ireland, Galway and Galway University Hospital, Galway, Ireland
3
Irish Meningitis and Sepsis Reference Laboratory, Temple Street Children’s University Hospital, Temple Street, Dublin, Ireland
*
Author to whom correspondence should be addressed.
Received: 15 January 2018 / Revised: 1 February 2018 / Accepted: 2 February 2018 / Published: 9 February 2018
(This article belongs to the Section Molecular Pathology, Diagnostics, and Therapeutics)
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Abstract

Bacterial meningitis infection is a leading global health concern for which rapid and accurate diagnosis is essential to reduce associated morbidity and mortality. Loop-mediated isothermal amplification (LAMP) offers an effective low-cost diagnostic approach; however, multiplex LAMP is difficult to achieve, limiting its application. We have developed novel real-time multiplex LAMP technology, TEC-LAMP, using Tth endonuclease IV and a unique LAMP primer/probe. This study evaluates the analytical specificity, limit of detection (LOD) and clinical application of an internally controlled multiplex TEC-LAMP assay for detection of leading bacterial meningitis pathogens: Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae. Analytical specificities were established by testing 168 bacterial strains, and LODs were determined using Probit analysis. The TEC-LAMP assay was 100% specific, with LODs for S. pneumoniae, N. meningitidis and H. influenzae of 39.5, 17.3 and 25.9 genome copies per reaction, respectively. Clinical performance was evaluated by testing 65 archived PCR-positive samples. Compared to singleplex real-time PCR, the multiplex TEC-LAMP assay demonstrated diagnostic sensitivity and specificity of 92.3% and 100%, respectively. This is the first report of a single-tube internally controlled multiplex LAMP assay for bacterial meningitis pathogen detection, and the first report of Tth endonuclease IV incorporation into nucleic acid amplification diagnostic technology. View Full-Text
Keywords: bacterial meningitis; nucleic acid diagnostics; loop-mediated isothermal amplification; multiplex; internal control bacterial meningitis; nucleic acid diagnostics; loop-mediated isothermal amplification; multiplex; internal control
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Higgins, O.; Clancy, E.; Cormican, M.; Boo, T.W.; Cunney, R.; Smith, T.J. Evaluation of an Internally Controlled Multiplex Tth Endonuclease Cleavage Loop-Mediated Isothermal Amplification (TEC-LAMP) Assay for the Detection of Bacterial Meningitis Pathogens. Int. J. Mol. Sci. 2018, 19, 524.

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