DESAIN DNA PELACAK SECARA IN SILICO SEBAGAI PENDETEKSI MUTASI PADA GEN embB Mycobacterium tuberculosis

  • Ade Ari Sundari, Udayana University Program Studi Farmasi, FMIPA, Universitas Udayana, Bukit Jimbaran, Bali-Indonesia, 80361
  • Ni Putu Monica Rosdiana Dewi Paramitha Program Studi Farmasi, FMIPA, Universitas Udayana, Bukit Jimbaran, Bali-Indonesia, 80361
  • Sagung Chandra Yowani Kelompok Studi MDR-TB & XDR-TB, FMIPA, Universitas Udayana, Bukit Jimbaran, Bali-Indonesia, 80361
  • Putu Sanna Yustiantara Kelompok Studi MDR-TB & XDR-TB, FMIPA, Universitas Udayana, Bukit Jimbaran, Bali-Indonesia, 80361

Abstract

ABSTRAK: Terapi lini pertama Tuberkulosis (TB) selalu menggunakan terapi kombinasi yaitu rifampisin, isoniazid, etambutol, dan pirazinamid. Penggunaan etambutol dalam terapi kombinasi TB digunakan untuk mencegah terjadinya resistensi terhadap obat lain namun tingkat resistensi etambutol secara bertahap meningkat. Mutasi pada operon embCAB bertanggung jawab pada resistensi etambutol dengan prevalensi tertinggi terjadi pada kodon 306 gen embB. Mutasi pada gen embB kodon 306 juga dikaitkan dengan adanya kecenderungan resistensi akibat peningkatan konsumsi obat sehingga dijadikan sebagai kandidat potential marker untuk board drug resistance, khususnya untuk MDR-TB. Penelitian ini akan mendesain urutan nukleotida TaqMan probe untuk mendeteksi mutasi M306I menggunakan program Clone Manager Suite 9.2. Hasil rancangan probe DNA kemudian dianalisis berdasarkan kriteria probe secara umum dan berdasarkan kriteria pelabelan TaqMan probe. Rancangan probe DNA mutan menggunakan program menghasilkan 10 probe yang memenuhi kriteria probe secara umum untuk mutasi M306I pada gen embB. Berdasarkan analisa pelabelan TaqMan probe, diperoleh 7 probe (E306MI4, E306MI5, E306MI6, E306MI7, E306MI9, E306MI10, dan E306MI13) untuk deteksi mutasi M306I pada gen embB. Hasil rancangan probe mutan yang telah memenuhi kriteria pelabelan TaqMan probe dapat digunakan untuk mendeteksi adanya mutasi kodon 306 gen embB Mycobacterium tuberculosis. Hasil perancangan TaqMan probe perlu diuji secara eksperimental untuk membuktikan efisiensi kerja dari probe tersebut agar dapat digunakan pada metode Real-Time PCR.


 


ABSTRACT: First-line Tuberculosis (TB) therapy always uses combination therapy, such as rifampicin, isoniazid, ethambutol, and pyrazinamide. Ethambutol was effective for preventing treatment failures caused by Mycobacterium tuberculosis isolates resistant to other anti-TB drugs however, the resistance rate of ethambutol has gradually increased. Mutations in the embCAB operon have been identi?ed to confer resistance to ethambutol, with embB codon 306 being the most frequently affected. embB306 mutations are associated a tendency for resistance due to increasing numbers of antibiotics consumption so it may be a potential marker for broad drug resistance, especially for MDR-TB. This research design the TaqMan probe nucleotide sequence for the M306I spesific mutation using the Clone Manager Suite 9.2 program. The results of the DNA probe design were then analyzed based on probe criteria in general and based on the TaqMan probe labeling criteria. The mutant DNA probes design using the program produced 10 probes thats have met the general probe criteria for the M306I mutation in the embB gene. Based on the TaqMan probe labeling analysis, theres 7 probes (E306MI4, E306MI5, E306MI6, E306MI7, E306MI9, E306MI10 and E306MI13) for the detection of M306I mutations in the embB gene. The results of the mutant probe design that has met the TaqMan labeling criteria can be used to detect mutations in M. tuberculosis embB gene codon 306. The results of the TaqMan probe need to be tested experimentally to prove the working efficiency of the probe so that it can be used in the Real-Time PCR method.

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References

[1] World Health Organization (WHO). 2018. Global Tuberculosis Report, World Health Organization, Geneva.
[2] Cheng, S., Cui, Z., Li, Y., and Hu, Z. 2014. Diagnostic Accuracy of a Molecular Drug Susceptibility Testing Method for the Antituberculosis Drug Ethambutol: a Systematic Review and Meta-Analysis, Journal of Clinical Microbiology, 52: 2913-2924.
[3] Moure, R., Español, M., Tudó, G., Vicente, E., Coll, P., Martin, J.G., Mick, V., Salvadó, M., and Alcaide, F. 2013. Characterization of the embB gene in Mycobacterium tuberculosis isolates from Barcelona and rapid detection of main mutations related to ethambutol resistance using a low-density DNA array. Journal of Antimicrobial Chemotherapy, 69 : 947-954
[4] Li, Y., Wang, Y., Zhang, Z., Gao, H., Wang, H., Cao, J., Zhang, S., Liu, Y., Lu, J., Xu, Z., and Daia, E., 2016. Association between embB Codon 306 Mutations, Phenotypic Resistance Profiles, and Genotypic Characterization in Clinical Mycobacterium tuberculosis Isolates from Hebei, China, Antimicrobial Agents and Chemotheramy, 60: 7295-7301.
[5] Mohammadi, B., Mohajeri, P., Rouhi, S., and Ramazanzadeh, R., 2018. The relationship between embB306 and embB406 mutations and ethambutol resistant in Mycobacterium tuberculosis isolated from patiens in west of Iran, Medical Journal of the Islamic Republic of Iran, 32: 1-4.
[6] Rezaei, F.M., Haeili, P., Mohajeri, A.H., Shahraki, Fooladi, A.I., Zahednamazi, F., Feizabadi, M., 2016, Frequency of mutational changes in the embB among the ethambutol resistant strains of Mycobacterium tuberculosis in Iran, The Journal of Infection in Developing Countries, 10: 363-368.
[7] Bakula, Z., Napiorkowska, A., Bielecki, J., Kopec, E.A., Zwolska, Z., and Jagielski, T., 2013. Mutations in the embB Gene and Their Association with Ethambutol Resistance in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates from Poland, BioMed Research International.
[8] Cordoba, B.C., Eusebio, D.M.J., Velasco, R.A., Salazar, R.M., Laborin, R.L., and Cuevas, R.Z., 2015. Mutation at embB Codon 306, a Potential Marker for the Identification of Multidrug Resistance Associated with Ethambutol in Mycobacterium tuberculosis, Antimicrobial Agents and Chemotherapy, 59: 5455-5462.
[9] Zhang, H., Chen, X., Wang, Z., Ren, Z., Wu, J., Sun, H. and Bai, X., 2015. Pyrosequencing analysis for mutations in embB codon306 among clinical Mycobacterium tuberculosis isolates from Qingdao, China. Int J Clin Exp Med. 8 : 11276-11282.
[10] Chou, C., Chen, C.H., Lee, T., and Peck, K., 2004. Optimization of probe length and the number of probes per gene for optimal microarray analysis of gene expression, Nucleic Acids Research, 32: 1-8
[11] McPherson, M and Moller, S., 2006. PCR Edisi 2, Taylor & Francis Group, New York.
[12] Navarro, E., Heras, G.S., Castano and Solera, J., 2015. Real-Time PCR Detection Chemistry, Clinica Chimica Acta, 439: 231-250.
[13] Zhao, L., Sun, Q., Liu, H., Xiao, T., Zhao, X., Li, G., Jiang, Y., Zeng, C., Wan, K., 2015. Analysis of embCAB Mutations Associated with Ethambutol Resistance in Multidrug-Resistant Mycobacterium tuberculosis Isolates from China, Antimicrobial Agents and Chemotherapy, 59: 2045-2050.
[14] Sun, Q., Xiao, T., Liu, H., Zhao, X., Liu, Z., Li, Y., Zeng, H., Zhao, L., and Wan, K., 2018. Mutations within embCAB Are Associated with Variable Level of Ethambutol Resistance in Mycobacterium tuberculosis Isolates from China, Antimicrobial Agents and Chemotherapy, 62: 1-8.
[15] Khosravi, A.D., Meghdadi, H., Ghadiri, A., Alami, A., Sina, A.H., Mirsaeidi, M., 2018. rpoB gene mutations among Mycobacterium tuberculosis isolates from extrapulmonary sites. Journal of Pathology, Microbiology and Immunology, 126: 241-247.
[16] Walker, J.M. dan Rapley, R., 2008. Medical Biomethods Handbook, Humana Press, Totowa, New Jersey.
[17] Alvandi, E., and Koohdani, F., 2014. Zip nucleic acid: a new reliable method to increase the melting temperature of real-time PCR probes, Journal of Diabetes & Metabolic Disorders, 13: 1-4.
[18] Dorak, M.T. 2006, Real-Time PCR, Taylor and Francis Group, New York.
[19] Anonim, 2006. Real-Time PCR Application Guide, Bio-Rad Laboratories Inc, USA.
[20] Walker, J.M. and Rapley, R., 2005. Medical Biomethods Handbook, Humana Press, Totowa, New Jersey.
[21] Bishop, J.L., Campbell, S. A., Farrell, P., Fitzgerald, M., Haugen, M., Kocmond, W., Madden, D. E., Murray, W. E., and Persing, D. H., 2015. Designing Real Time Assays on the SmartCycler® II System, Cepheid Technical Support, United States
[22] Borah, P., 2011. Primer Designing for PCR, Science Vision, 11 : 134-136.
[23] Butler, J.M. 2011, Advanced topics in forensic DNA typing: methodology, Academic press, USA.
[24] Johansson, M.K., 2006. Choosing Reporter-Ouencher Pairs for Efficient Quenching Through Formation of Intramolecular Dimers, Methods in Molecular Biology, 335: 17-29.
[25] Murray, J. L., Hu, P., dan Shafer D. A., 2014. Seven Novel Probe System for RealTime PCR Provide Absolute Single-Base Discrimination, Higher Signaling, and Generic Components, The Journal of Molecular Diagnostics, 16: 627-638.
[26] Nazarenko, I., Pires, R., Lowe, B., Obaidy, M., dan Rashtchian, A., 2002. Effect of Primary and Secondary Structure of Oligodeoxyribonucleo- tides on The Fluorescent Properties of Conjugated Dyes. Nucleic Acids Research, 30: 2089-2195.
[27] Didenko, V. V. 2006. Fluorescent Energy Transfer Nucleic Acid Probe Design and Protocols. Humana Press Inc, Totowa, New Jersey. 17-80.
[28] Livak, K. J., Flood S. J. A., Marmaro, J., Giusti, W., dan Deetz, K., 1995. Oligonucleotides With Fluorescent Dyes At Opposite Ends Provide A Quenched Probe System Useful For Detecting PCR Product and Nucleic Acid Hybridization. Genome Research. 4: 357-362.
[29] Marras, S.A., 2006. ‘Selection of fluorophore and quencher pairs for fluorescent nucleic acid hybridization probes’ in Didenko, Fluorescent energy transfer nucleic acid probes, Humana Press, Totowa, pp 3-16.
Published
2020-05-31
How to Cite
SUNDARI, Ade Ari et al. DESAIN DNA PELACAK SECARA IN SILICO SEBAGAI PENDETEKSI MUTASI PADA GEN embB Mycobacterium tuberculosis. CAKRA KIMIA (Indonesian E-Journal of Applied Chemistry), [S.l.], v. 8, n. 1, p. 41 - 50, may 2020. ISSN 2302-7274. Available at: <https://ojs.unud.ac.id/index.php/cakra/article/view/62804>. Date accessed: 07 oct. 2022.