ISOLASI DNA METAGENOMIK DARI SPUTUM PASIEN TUBERKULOSIS DAN AMPLIFIKASI DENGAN PRIMER PROMOTER inhA

  • Ni Made Yustikarini Program Studi Kimia Terapan , Program Pasca Sarjana Universitas Udayana Bali Indonesia
  • Sagung Chandra Yowani 1. Program Studi Kimia Terapan , Program Pasca Sarjana Universitas Udayana Bali Indonesia 2. Kelompok Studi MDR & XDR TB FMIPA Universitasa Udayana Bali Indonesia
  • I Nengah Wirajana 1. Program Studi Kimia Terapan , Program Pasca Sarjana Universitas Udayana Bali Indonesia 2. Kelompok Studi MDR & XDR TB FMIPA Universitasa Udayana Bali Indonesia

Abstract

 

ABSTRAK: Penelitian ini bertujuan untuk memperoleh DNA metagenomik dari sputum pasien tuberkulosis dan mengamplifikasi dengan menggunakan primer promoter inhA. Penelitian ini dilakukan dalam tiga tahap, yaitu: isolasi DNA metagenomik dari sputum pasien tuberkulosis, amplifikasi menggunakan sepasang primer promoter inhA dari M. tuberculosis dengan metode PCR, dan elektroforesis gel agarosa terhadap hasil amplifikasi. Elektroforegram hasil amplifikasi menunjukkan bahwa isolasi DNA metagenomik dari sputum pasien tuberkulosis telah berhasil dilakukan dengan metode modifikasi maupun dengan kit. Ukuran pita fragmen DNA sekitar 284 bp dari hasil amplifikasi (amplikon) yang diperoleh dari DNA metagenomik sputum P.48B, P.46B, dan P.47B  MDR- TB  merupakan ukuran yang sesuai dengan bagaian dari daerah promoter inhA M. tuberculosis. Ukuran amplikon ini sama dengan ukuran amplikon yang sebelumnya telah diperoleh dari amplifikasi terhadap DNA M. tuberculosis oleh peneliti sebelumnya dengan menggunakan primer yang sama.

ABSTRACT: The aims of this research were to obtain metagenomic DNA from sputum of tuberculosis patients and to amplify it by using inhA promoter primer. This research was conducted in three steps covering the DNA metagenomic isolation from sputum of tuberculosis patients, amplification of inhA promoter region of M. tuberculosis by using specific primer pair by PCR (Polymerase Chain Reaction) method, and electrophoresis of amplified products using agarose gel. The electrophoregram of amplified products showed that the metagenomic DNA isolation from sputum of tuberculosis patients was successfully carried out both by the modified method and by a kit. The size of DNA fragment bands about 284 bp of amplified products (amplicons) which obtained from the metagenomic DNAs of P.48B, P.46B, P.47B sputum was suitable size with a part of inhA promoter region of M. tuberculosis. The size of these amplicons was the same size with the amplicons from M. tuberculosis DNA reported by other researchers.

Downloads

Download data is not yet available.

References

[1] Sabree, Z.L., Rondon, M.R., and Handels- man, J. 2009. Metagenomics. Elsevier 622-632.
[2] Kim W. 2012, Application of Metage- nomic Techniques: Understanding the Unrevealed Human Microbiota and Explaining the ini Clinical Infectious Disease. Journal of Bacteriology and Virology 42(4): 263-275.
[3] Miller, R.R., Montoya, V., Gardy, J.L., Patrick, D.M, Tang P 2013. Metagenomics for pathogen detection in public health. Genome Medicine 5:81
[4] Weinstock G.M, 2012. Genomic approach- es to studying the human microbiota. Nature 489 : 250-256
[5] Nakamura, S., Maeda, N., Miron, I.M., Yoh, M., Izutsu, K., Kataoka, C., Honda, T., Yasunaga, T., Nakaya, T., Kawai, J., Hayashizaki, Y., Horii, T., and Iida, T. 2008. Metagenomic Diagnosis of Bacterial Infections. Emerging Infectious Diseases (www.cdc.gov/eid). 14(11) : 1784-1786.
[6] Zhang M, Yue J, Yang YP, Zhang HM, Lei JQ, Jin RL, 2005. Detection of mutations associated with isoniazid resistance in Mycobacterium tuberculosis isolates from China. J. Clin. Microbiol. 43: 5477-82.
[7] Heifets LB, Barnes PF. 1994 Current laboratory methods for the diagnosis of tuberculosis. In: Bloom BR,editor. Tuberculosis, pathogenesis, protection, andcontrol. Washington DC: American Society for Microbiology. p. 85-110.
[8] Kent,P.T and Kubica,G.P 1985. A Guide gor The Level III Laboratory. Centres for Disease and Control, Atlanta,Ga.
[9] Kirschner,P., Springer,B., Vogel,U., et al., 1993. Genotypic identification of mycobacteria by nucleic acid sequence determination, report of a 2-year experience in a clinical laboratory. J.Clin.Microbiol., 31: 2882-2889
[10] Zhou, J., Bruns, M. A., & Tiedje, J. M., 1996. DNA recovery from soils of diverse composition. Applied and Environmental Microbiology 62: 316–322.
[11] Patel, S., Yates, M., and Saunders, N.A., 1997. PCR-Enzyme-Linked Immuno- sorbent Assay and Partial rRNA Gene Sequencing: a Rational Approach to Identifying Mycobacteria. J. Clin. Microbiol. 35:2375-2380.
[12] Chen, X., F. Kong., Q. Wang., C. Li., J. Zhang, dan G. L. Gilbert. 2011. Rapid Detection of Isoniazid, Rifampin, and Ofloxacin Resistance in Mycobacterium tuberculosis Clinical Isolates Using High-Resolution Melting Analysis. Journal of Clinical Microbiology 49: 3450-3457.
[13] McPherson, M.J. and Moller, S.G., 2006. PCR 2nd ed. UK: Taylor and Francis Group.
Published
2015-10-28
How to Cite
YUSTIKARINI, Ni Made; YOWANI, Sagung Chandra; WIRAJANA, I Nengah. ISOLASI DNA METAGENOMIK DARI SPUTUM PASIEN TUBERKULOSIS DAN AMPLIFIKASI DENGAN PRIMER PROMOTER inhA. CAKRA KIMIA (Indonesian E-Journal of Applied Chemistry), [S.l.], v. 3, n. 3, p. 56-62, oct. 2015. ISSN 2302-7274. Available at: <https://ojs.unud.ac.id/index.php/cakra/article/view/20281>. Date accessed: 07 apr. 2020.
Section
Articles

Keywords

Metagenomic DNA, sputum, Mycobacterium tuberculosis, inhA promoter