Evaluating the use of loop-mediated isothermal amplification (LAMP) method for detection of <em>Mycobacterium tuberculosis</em> in Indonesian clinical isolates
DOI:
https://doi.org/10.13181/mji.v21i4.502Keywords:
Loop-mediated isothermal amplification, rim gene, 16S rRNA gene, gyrB gene, Mycobacterium tuberculosisAbstract
Background: Loop-mediated isothermal amplification (LAMP) is a method already claimed as a simple technique to amplify DNA/ RNA using four to six primers as "a set" from conserved sequence of target gene. In this study we optimize the use of LAMP for detection of Mycobacterium tuberculosis in clinical isolates from Indonesia.
Methods: Procedures to perform LAMP were optimized, then the method was applied to 122 archieved samples of DNA's Mtb from clinical TB patients with Acid Fast Bacilli (AFB) smears positive. The samples were obtained in 2008 from 13 provinces in Indonesia for genotyping study, which then become collections of Center for Biomedical and Basic Technology of Health (CBBTH), NIHRD Indonesia. The optimization tests include sensitivity and specificity tests of several sets primers, which were evaluated using 10-fold serially diluted DNA of Mtb H37Rv and 12 species of Mycobacteria. Three equipments consisted of LAMP turbidimeter, heating block and water bath were compared for its ability in DNA amplification. Detection of M. tuberculosis from clinical isolates used set primers specific for gyrB gene, amplicon was detected with UV fluorescence system.
Results: The results showed that the highest sensitivity was obtained using the set primers specific for 16S rRNA and gyrB which could detect 10.0 fg to 1.0 pg genomic DNA of Mtb H37Rv. The set primers specific for gyrB gene was the most specific primers. Application of LAMP using gyrB set primers on Indonesian clinical isolates showed 94.2% (114/121) positivity rate.
Conclusion: LAMP method is potentially used in TB diagnosis in Indonesia. (Med J Indones. 2012;21:188-95)
Downloads
References
World Health Organization. New technologies for tuberculosis control: a framework for their adoption, introduction and implementation. France: WHO Library Cataloguing-in-Publication Data; 2007.
Forbes BA, Sahm DF, Weissfeld AS. Bailey & Scott'?s diagnostic microbiology. 12th ed. Philadelphia: Mosby Elsevier; 2007. p. 509-478.
Notomi T, Okayama H, Masubuchi H, Yonekawa T, Watanabe K, Amino N, et al. Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 2000;28:e63. https://doi.org/10.1093/nar/28.12.e63
Nagamine K, Watanabe K, Ohtsuka K, Hase T, Notomi T. Loop-mediated isothermal amplification reaction using a nondenatured template. Clin Chem. 2001;47(9):1742-3. https://doi.org/10.1093/clinchem/47.9.1742
Iwamoto T, Sonobe T, Hayashi K. Loop-mediated isothermal amplification for direct detection of Mycobacterium tuberculosis complex, M. avium, and M. intracellulare in sputum samples. J Clin Microbiol. 2003;41:2616-22. https://doi.org/10.1128/JCM.41.6.2616-2622.2003
Kuboki N, Inoue N, Sakurai T, Di Cello F, Grab DJ, Suzuki H, et al. Loop-mediated isothermal amplification for detection African trypanosomes. J Clin Microbiol. 2003;41:5517-24. https://doi.org/10.1128/JCM.41.12.5517-5524.2003
Hong TC, Mai QL, Cuong DV, Parida M, Minekawa H, Notomi T, et al. Development and evaluation of a novel loop-mediated isothermal amplification method for rapid detection of severe acute respiratory syndrome coronavirus. J Clin Microbiol. 2004;42:1956-61. https://doi.org/10.1128/JCM.42.5.1956-1961.2004
Hase, T. Rapid detection of Mycobacterium tuberculosis complex from sputum samples using novel loop-mediated isothermal amplification. EIKEN Chemical Co., LTD; 2007.
Ihira M, Yoshikawa T, Enomoto Y, Akimoto S, Ohashi M, Suga S, et al. Rapid diagnosis of human herpesvirus 6 infection by a novel DNA amplification method, loop-mediated isothermal amplification. J Clin Microbiol. 2004;42(1):140-5. https://doi.org/10.1128/JCM.42.1.140-145.2004
EIKEN Genome Site [Internet]. 2007 [cited 2009 Nov]. Available from: http://loopamp.eiken.co.jp/e/lamp/index.html. 2009/11/25.
Boehme CC, Nabeta P, Henostroza G, Raqib R, Rahim Z, Gerhardt M, et al. Operational feasibility of using loop-mediated isothermal amplificatin for diagnosis of pulmonary tuberculosis in microscopy centers of developing countries. J Clin Microbiol. 2007;45:1936-40. https://doi.org/10.1128/JCM.02352-06
Parida M, Posadas G, Inoue S, Hasebe F, Morita K. Real-time reverse transcription loop-mediated isothermal amplification for rapid detection of West Nile virus. J Clin Microbiol. 2004;42:257-63. https://doi.org/10.1128/JCM.42.1.257-263.2004
Conville PS, Witebsky FG. Analysis of multiple differing copies of the 16S rRNA gene in five clinical isolates and three type strains of Nocardia species and implications for species assignment. J Clin Microbiol. 2007;45:1146-51. https://doi.org/10.1128/JCM.02482-06
Pandey BD, Poudel A, Yoda T, Tamaru A, Oda N, Fukushima Y, et al. Development of an in-house loop-mediated isothermal amplification (LAMP) assay for detection of Mycobacterium tuberculosis and evaluation in sputum samples of Nepalese patients. J Med Microbiol. 2008;57:439-43. https://doi.org/10.1099/jmm.0.47499-0
Zhu RY, Zhang KX, Zhao MQ, Liu YH, Xu YY, Ju CM, et al. Use of visual loop-mediated isothermal amplification of rimM sequence for rapid detection of Mycobacterium tuberculosis and Mycobacterium bovis. J Microbiol Methods. 2009;78:339-43. https://doi.org/10.1016/j.mimet.2009.07.006
Yamaguchi, et al. in Japanese alphabeth. 2006. [cited 2009 Dec]. Available from: http://www.j-tokyo.com/2006/C12N/JP2006-061134.shtml. Japanese.
Wu X, Zhang J, Liang J, Lu Y, Li H, Li C, et al. Comparison of three methods for rapid identification of mycobacterial clinical isolates to the species level. J Clin Microbiol. 2007;45(6):1898-903. https://doi.org/10.1128/JCM.02253-06
Chimara E, Ferrazoli L, Leao SC. Mycobacterium tuberculosis complex differentiation using gyrB restriction fragment length polymorphism analysis. Mem Inst Oswaldo Cruz, Rio de Janeiro. 2004;99(7):745-8. https://doi.org/10.1590/S0074-02762004000700014
Downloads
Published
How to Cite
Issue
Section
License
Authors who publish with Medical Journal of Indonesia agree to the following terms:
- Authors retain copyright and grant Medical Journal of Indonesia right of first publication with the work simultaneously licensed under a Creative Commons Attribution-NonCommercial License that allows others to remix, adapt, build upon the work non-commercially with an acknowledgment of the work’s authorship and initial publication in Medical Journal of Indonesia.
- Authors are permitted to copy and redistribute the journal's published version of the work non-commercially (e.g., post it to an institutional repository or publish it in a book), with an acknowledgment of its initial publication in Medical Journal of Indonesia.
