PCR Multipleks untuk Identifikasi Mycobacterium tuberculosis Resisten terhadap Isoniazid dan Rifampisin pada Galur Lokal Balai Laboratorium Kesehatan Provinsi Jawa Barat

Iman Permana Maksum, Suhaili Suhaili, Rizki Amalia, Dian Siti Kamara, Saadah Dian Rachman, Rifky W. Rachman


The incidence of multidrug-resistant tuberculosis (MDR-TB) cases has become the biggest source of the problem in the effort to eradicate tuberculosis (TB) disease in Indonesia. MDR-TB is a resistant TB bacteria to the two, at least, first-line TB drugs, e.g., rifampin and isoniazid. Unfortunately, the current diagnostics methods to identify the MDR-TB are still slow, unspecific, and inaccurate. The purpose of this study is to identify the isoniazid- and rifampin-resistant M. tuberculosis (local strain Balai Laboratorium Kesehatan Provinsi Jawa Barat) by using multiplex PCR method. The TB bacteria colonies were cultivated in Middlebrook 7h9 broth media, which followed by the isolation of chromosomal DNA. The best PCR condition was achieved by optimizing the annealing temperature, the concentration of magnesium chloride, and a number of the cycle. Multiplex PCR was conducted with inhA1-inhA2, rpoB1- rpoB2, katG1- katG2, and B1-B2 pair primers. Furthermore, the PCR product was characterized on 2% gel agarose electrophoresis which stained by using ethidium bromide. The result showed that isoniazid- and rifampin-resistant M. tuberculosis sample could be identified using multiplex PCR, producing DNA fragments with a size of 71 bp, 124 bp 186 bp, and 200 bp. A non-MDR-TB only produced one DNA fragments with a size of 200 bp. Therefore, it can be concluded that MDR-TB and non-MDR-TB can be distinguished using multiplex PCR with a combination of four pair primers.




M. tuberculosis, MDR-TB, multiplex PCR


Bachmann L, Däubl B, Lindqvist C, Kruckenhauser L, Teschler-Nicola M, Haring E. 2008. PCR diagnostics of Mycobacterium tuberculosis in historic human long bone remains from 18th century burials in Kaiserebersdorf, Austria. BMC Research Notes. 1: 1–6. https://doi.org/10.1186/1756-0500-1-83.

Collins D, Hafidz F, Mustikawati D. 2017. The economic burden of tuberculosis in Indonesia. The International Journal of Tuberculosis and Lung Disease, 21(9), 1041–1048. https://doi.org/10.5588/ijtld.16.0898.

Fessenden R, Fessenden J. 1992. Kimia Organik Jilid 1. Jakarta(ID): Erlangga.

Herrera-Leon L, Jimenez M, Molina T, Saiz P, Saez-Nieto J. 2005. New multiplex PCR for rapid detection of isoniazid-resistant Mycobacterium tuberculosis clinical isolates. Antimicrobial Agents and Chemotherapy, 49(1): 144–147. https://doi.org/10.1093/nar/15.3.1311

Kolyva AS, Karakouis PC. 2016. Old and new drugs : mechanism of action and resistance. Intechopen. 9(10): 209–232. https://doi.org/10.5772/711.

Li J, Xin J, Zhang L, Jiang L, Cao H, Li L. 2012. Rapid detection of rpoB mutations in rifampin resistant M. tuberculosis from sputum samples by denaturing gradient gel electrophoresis. International Journal of Medical Sciences. 9(2): 148–156. https://doi.org/10.7150/ijms.3605.

Maksum I. 2017. PCR Dalam Investigasi Penyakit Mitokondria. Jatinangor (ID): Alqaprint, Cakrawala Baru Dunia Buku.

Maksum IP, Sriwidodo, Gaffar S, Hassan K, Subroto T, Soemitro S. 2017. Teknik Biologi Molekular. (AA Wildan, Ed.) (1st ed.). Jatinangor (ID): Alqaprint Jatinangor.

Sandegren L, Groenheit R, Koivula T, Ghebremichae S, Advani A, Castro E, Källenius G. 2011. Genomic stability over 9 years of an isoniazid resistant mycobacterium tuberculosis outbreak strain in Sweden. PLoS ONE, 6(1): https://doi.org/10.1371/journal.pone.0016647.

Seifert M, Catanzaro D, Catanzaro A, Rodwell TC. 2015. Genetic mutations associated with isoniazid resistance in Mycobacterium tuberculosis: A systematic review. PLoS ONE, 10(3): 1–13. https://doi.org/10.1371/journal.pone.0119628.

Shimizu Y, Dobashi K, Yoshikawa Y, Yabe S, Higuchi S, Koike Y, Mori M. 2008. Five-antituberculosis drug-resistance genes detection using array system. Journal of Clinical Biochemistry and Nutrition, 42(3), 228–34. https://doi.org/10.3164/jcbn.2008033.

Smith T, Wolff K, Nguyen L. 2014. Molecular Biology of Drug Resistance in Mycobacterium tuberculosis. 374: 53–80.

Stagg HR, Lipman MC, McHugh TD, and J. H. (2017). Isoniazid resistant tuberculosis- a cause for concern. Int J.T. Uberc Lung Dis. 21(2): 129–139. https://doi.org/10.5588/ijtld.16.0716.Isoniazid

Tambunan USF, Sugito S, Parikesit AA. 2010. Design and Evaluation of Three Pair Primers for Exon 1 Amplification of Hyaluroglucosaminidase-1 Gene. OnLine Journal of Biological Sciences. 10(2): 66–72.

Tseng ST, Tai CH, Li CR, Lin CF, Shi ZY. 2015. The mutations of katG and inhA genes of isoniazid-resistant Mycobacterium tuberculosis isolates in Taiwan. Journal of Microbiology, Immunology and Infection, 48(3): 249–255. https://doi.org/10.1016/j.jmii.2013.08.018

Tu YK, Hong YY, Chen YC. 2009. Finite element modeling of kirschner pin and bone thermal contact during drilling. Life Science Journal. 6(4): 23–27. https://doi.org/10.1007/82

WHO. 2018. Tubercolusis (Tb) Seventy-first World Health Assembly commits to accelerate action to End TB. Retrieved from http://www.who.int/tb/en/.

Full Text: PDF

DOI: 10.15408/jkv.v4i2.7226


  • There are currently no refbacks.

Copyright (c) 2019 Iman Permana Maksum, Suhaili Suhaili, Rizki Amalia, Dian Siti Kamara, Saadah Dian Rachman, Rifky W. Rachman

Creative Commons License
This work is licensed under a Creative Commons Attribution-ShareAlike 4.0 International License.