Evaluation of an in-house loop-mediated isothermal amplification assay targeting the gene sequence IS6110 for the detection of mycobacterium tuberculosis in clinical samples

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Evaluation of an in-house loop-mediated isothermal amplification assay targeting the gene sequence IS6110 for the detection of mycobacterium tuberculosis in clinical samples

 

Author: Kua, Zeigmund Dean Alexander Chan
Title: Evaluation of an in-house loop-mediated isothermal amplification assay targeting the gene sequence IS6110 for the detection of mycobacterium tuberculosis in clinical samples
Degree: M.Sc.
Year: 2015
Subject: Mycobacterium tuberculosis -- Diagnosis
Gene amplification.
Hong Kong Polytechnic University -- Dissertations
Department: Dept. of Health Technology and Informatics
Pages: xviii, 112 leaves : illustrations (some color) ; 30 cm
Language: English
InnoPac Record: http://library.polyu.edu.hk/record=b2800047
URI: http://theses.lib.polyu.edu.hk/handle/200/7905
Abstract: Tuberculosis (TB) is a common human disease caused by Mycobacterium tuberculosis. Even though M. tuberculosis was discovered more than a century ago, the burden of TB is still felt throughout the world. An early diagnosis of M. tuberculosis is crucial for both the treatment of the patient and the control of TB in the community. However, Identification of M. tuberculosis has been a problem for clinical laboratories, due to their slow growth rate. Conventional culture-based methods that are being used in the laboratory have limitations on speed, specificity or sensitivity. Molecular tests such as real-time PCR on the other hand have the drawback of being expensive. Therefore there is a need to develop a test that is rapid, highly sensitive and highly specific while also being cost-effective. In recent years, a novel technique for nucleic acid amplification method known as loop-mediated isothermal amplification assay has been developed to address this concern. The key innovative feature of the LAMP assay is that amplification of DNA sequences is done in isothermal conditions, in which no change of temperature is required. This key feature of LAMP assay eliminates the need to use specialized equipment like “thermal cyclers that can rapidly change thermal conditions, this makes LAMP assay inexpensive. This study aims to evaluate an in-house LAMP assay for the detection of M. tuberculosis in clinical samples. Three sets of oligonucleotide primers targeting the gene sequence IS6110 specific to M. tuberculosis complex were designed. Optimization of the LAMP reaction was done. A total of 292 clinical respiratory samples and 30 bacterial cultures were used to study the LAMP assay. Analytical sensitivity of the LAMP assay was found to be 100 CFU/mL for sputum specimen, while analytical specificity was virtually 100%. The LAMP assay targeting IS6110 successfully detected M. tuberculosis in clinical respiratory samples from patients with TB. The diagnostic sensitivity of the LAMP assay for true positive samples was 91.4% (53/58), with specificity for true negative samples of 99.6% (233/234). In this regard, LAMP assay targeting the gene sequence IS6110 is rapid with high sensitivity and specificity and at the same time cost effective method for the early detection of M. tuberculosis in clinical samples.

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