Evaluation of an internally controlled multiplex tth endonuclease cleavage loop-meditated isothermal amplification (tec-lamp) assay for the detection of bacterial meningitis pathogens
Higgins, Owen Clancy, Eoin Cormican, Martin Boo, Teck Cunney, Robert Smith, Terry
Higgins, Owen
Clancy, Eoin
Cormican, Martin
Boo, Teck
Cunney, Robert
Smith, Terry
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Publication Date
2018-02-09
Keywords
bacterial meningitis, nucleic acid diagnostics, loop-mediated isothermal amplification, multiplex, internal control, real-time pcr, influenzae type-b, haemophilus-influenzae, neisseria-meningitidis, streptococcus-pneumoniae, salmonella-enterica, clinical validation, assimilating probes, rapid detection, diagnosis
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journal article
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Citation
Higgins, Owen; Clancy, Eoin; Cormican, Martin; Boo, Teck; Cunney, Robert; Smith, Terry (2018). Evaluation of an internally controlled multiplex tth endonuclease cleavage loop-meditated isothermal amplification (tec-lamp) assay for the detection of bacterial meningitis pathogens. International Journal of Molecular Sciences 19 (2),
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.
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Publisher
MDPI AG