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

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...

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Bibliographic Details
Published in:International journal of molecular sciences 2018-02, Vol.19 (2), p.524
Main Authors: Higgins, Owen, Clancy, Eoin, Cormican, Martin, Boo, Teck Wee, Cunney, Robert, Smith, Terry J
Format: Article
Language:English
Subjects:
Assaying
Bacteria
bacterial meningitis
Diagnostic systems
Endonuclease
Genomes
internal control
loop-mediated isothermal amplification
Meningitis
Morbidity
multiplex
Multiplexing
nucleic acid diagnostics
Nucleic acids
Pathogens
Real time
Streptococcus infections
Streptococcus pneumoniae
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Summary: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 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: , and . 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 , and 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 endonuclease IV incorporation into nucleic acid amplification diagnostic technology.
ISSN:1422-0067
1661-6596
1422-0067
DOI:10.3390/ijms19020524