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Title: Development and clinical validation of a loop mediated isothermal amplification (LAMP) method for the rapid detection of Neisseria meningitidis
Author: McKenna , James Patrick
Awarding Body: Queen's University Belfast
Current Institution: Queen's University Belfast
Date of Award: 2013
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Loop mediated isothermal amplification (LAMP) is an innovative technique which allows the rapid detection of target nucleic acid sequences under isothermal conditions without the need for complex instrumentation. The development, systematic optimisation and clinical validation of a LAMP assay targeting the ctrA gene for the rapid detect ion of capsular N. meningitidis is described. Highly specific detection of capsular N. meningitidis type strains and clinical isolates was demonstrated, with no cross reactivity with other Neisseria spp., or with a comprehensive panel of other common human pathogens. The lower limit of detection was determined as 6 ctrA gene copies detectable in 48 minutes, with positive reactions readily identifiable visually via a simple colour change. High copy numbers could be detected in 22 minutes. Clinical Validation was carried out by retrospectively and prospectively analysing a total of 935 clinical specimens from a total of 603 patients. The level of agreement between the ctrA LAMP assay and a confirmatory test result (Culture, MRU PCR, ctrA RT-PCR) for each patient was used to evaluate the performance of LAMP for the detection of capsular N. meningitidis. The overall sensitivity and specificity of the designed ctrA LAMP assay was determined as 94.6% and 99.6% with positive and negative predictive values of 96.4% and 99.5% respectively. The potential utility of a positive ctrA LAMP respiratory specimen result to indicate MD was investigated. The sensitivity and specificity of respiratory• specimens in this regard was 89.3% and 97.7% with positive and negative predictive values of 80.7% and 98.9% respectively. Rapid LAMP testing of non-invasive respiratory specimens has significant potential to accelerate the diagnosis of MD. The LAMP method represents a simple, rapid, sensitive, and highly specific technique for the detection of N. meningitidis, and has the potential to be used as a POC molecular test and in resource poor settings.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available