Evaluating the use of loop-mediated isothermal amplification (LAMP) method for detection of Mycobacterium tuberculosis in Indonesian clinical isolates

  • Vivi Lisdawati
  • Tomohiro Oshibe
  • Hidetaka Tsuji
  • Tjahjani M. Sudiro
  • Myrna Adianti
  • Triyani Sukarso
  • Holy Arief
  • Hak Hotta
  • Pratiwi Sudarmono
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Abstract

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 âa setâ 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 DNAâs 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)

Keywords: Loop-mediated isothermal amplification, rim gene, 16S rRNA gene, gyrB gene, Mycobacterium tuberculosis

Author Biographies

Vivi Lisdawati
Center for Biomedical and Basic Technology of Health, National Institute of Health Research and Development, Ministry of Health, Jakarta, Indonesia
Tomohiro Oshibe
Hyogo Perfectural Institute of Public Health and Consumer Science, Public Health Science Research, Japan
Hidetaka Tsuji
Hyogo Perfectural Institute of Public Health and Consumer Science, Public Health Science Research, Japan
Tjahjani M. Sudiro
Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia
Triyani Sukarso
Department of Microbiology, Kobe University Graduate School of Medicine, Japan
Holy Arief
Center for Biomedical and Basic Technology of Health, National Institute of Health Research and Development, Ministry of Health, Jakarta, Indonesia
Hak Hotta
Department of Microbiology, Kobe University Graduate School of Medicine, Japan
Pratiwi Sudarmono
Department of Microbiology, Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia

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Published
2012-11-01
How to Cite
1.
Lisdawati V, Oshibe T, Tsuji H, Sudiro TM, Adianti M, Sukarso T, Arief H, Hotta H, Sudarmono P. Evaluating the use of loop-mediated isothermal amplification (LAMP) method for detection of <em>Mycobacterium tuberculosis</em&gt; in Indonesian clinical isolates. Med J Indones [Internet]. 2012Nov.1 [cited 2020Oct.28];21(4):188-95. Available from: http://mji.ui.ac.id/journal/index.php/mji/article/view/502
Section
Basic Medical Research

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