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BioMed Research International
Volume 2013 (2013), Article ID 926230, 6 pages
http://dx.doi.org/10.1155/2013/926230
Research Article

Detection of Mycobacterium tuberculosis by Using Loop-Mediated Isothermal Amplification Combined with a Lateral Flow Dipstick in Clinical Samples

1Innovative Learning Center, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand
2Centex Shrimp, Faculty of Science, Mahidol University, Bangkok 10400, Thailand
3National Center for Genetic Engineering and Biotechnology (BIOTEC), National Science and Technology Development Agency, Pathumthani 12120, Thailand
4Department of Pathology, Faculty of Medicine, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand
5Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Sukhumvit 23, Bangkok 10110, Thailand

Received 3 November 2012; Revised 2 February 2013; Accepted 5 February 2013

Academic Editor: Mina Hur

Copyright © 2013 Thongchai Kaewphinit et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Tuberculosis (TB) is a communicable disease caused by the bacterium Mycobacterium tuberculosis (MTB) and is a persistent problem in the developing countries. Loop-mediated isothermal amplification (LAMP) allows DNA to be amplified rapidly at a constant temperature. Here, a LAMP method was combined with a chromatographic lateral-flow dipstick (LFD) to detect IS6110 gene of M. tuberculosis specifically and rapidly. The reaction was optimized at 63°C for 60 min, and the amplified DNA hybridized to an FITC-labeled oligonucleotide probe for 5 min was detected at the LFD test line 5 min after application. Excluding the step of DNA extraction, the test results could be generated approximately within 1 h. In addition to the advantage of short assay time, this technique could avoid the contact of carcinogenic ethidium bromide due to the exclusion of the electrophoresis analysis step. Furthermore, the data indicated that LAMP-LFD could detect M. tuberculosis genomic DNA as little as 5 pg. The technique showed a significant specificity since no cross-hybridization to M. intracellulare (MIC), M. fortuitum (MFT), M. avium (MAV), M. kansasii (MKS), and M. gordonae (MGD) genomic DNAs was observed. In the clinical unknown samples test, the sensitivity of LAMP-LFD was 98.92% and the specificity was 100% compared to those of the standard culture assay. Based on its sensitivity, specificity, rapidity, low cost, and convenience, LAMP-LFD could be applicable for use in both laboratories and epidemiological surveys of MTB.