Authors:

I Gusti A. A. Santhi Rahmaryani, Ni Kadek Ariani, Dyah Subadrika Warma Dewi, Ni Komang Sasi Ani, Ade Ari Sundari, Kadek Widya Yuli Hartati, Sagung Chandra Yowani

Abstract:

“High-level resistance to isoniazid as a first-line tuberculosis drugs can be caused by mutations in codon 315 katG Mycobacterium tuberculosis . Mutation at codon 315 is the most frequent mutation with the highest amino acid variation, compared to other codons in the Mycobacterium tuberculosis katG gene. Therefore, a specific probe is required for rapid and proper detection of mutations at codon 315. In this study, the design of a nucleotide sequence probe with TaqMan labeling was performed using Clone Manager Suite 6 software . The mutant probe obtained was analysed in two stages. The initial analysis is based on the length of the probe (22-30 bases), Tm (70ºC), %GC (35-65%), not in hairpin form, dimer (< 5 bases), runs and repeat (? 4 for base A, T, C, and < 3 for base G). Furthermore the final analysis was carried out with no G base in 2 bases at the end of the 5’ probe and the amount of base C ? G. The study resulted in 260 probe mutants. After the initial analysis, 11 mutant probes were obtained to recognize mutations in the codon of 315 katG Mycobacterioum tuberculosis genes. The probe consists of 2 probes for the S315T mutation, 6 probes for S315N mutation, and 3 probes for S315V mutation. The criteria of the 11 mutant probes are 22-23 bases long, Tm 70ºC, % GC 56-63%, 4 dimer , 2 runs , and does not have repeats and does not form hairpin at a temperature of 56ºC. Based on the final analysis, 3 mutant probes were obtained fulfilling the TaqMan probe labeling criteria, namely K315MT1 for specific detection of mutation S?T and K315MN5, then K315MN23 for specific detection of mutation S?N. The conclusion of this study shows that the best mutant nucleotide sequence probes for the detection of mutations at codon of 315 KatG Mycobacterium tuberculosis genes are 5’-FAM-CC ACC GGC ATC GAG GTC GTA TG-TAMRA-3’; 5’FAM-ATC ACC AAC GGC ATC GAG GTC G-TAMRA-3’; dan 5’FAM-C ACC AAC GGC ATC GAG GTC GTA T-TAMRA-3’. The design of the mutant probe according to the TaqMan probe criterion for real-time PCR was obtained by 3 probes from the 11 selected mutant probes in the initial analysis.”

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References

  • [1] World Health Organization(WHO). 2014. Global
  • Tuberculosis Report 2014. Perancis: World Health
  • Organization.
  • [2] World Health Organization(WHO). 2015. Global
  • Tuberculosis Report 2015. Perancis: World Health
  • Organization.
  • [3] Lisdawati, V., N. Puspandari, L. Rif’ati, T. Soekarno,
  • M, Melatiwati, K. Syamsidar, L. Ratnasari, N.
  • Izzatun, dan I. Parwati. 2015. Molecular
  • Epidemiology Study of Mycobacterium tuberculosis
  • and Its Susceptibility to Anti-TuberculosisDrugs in
  • Indonesia. BMC Infectious Disease. Volume 15: 366-
  • 373.
  • [4] Babamahmoodi, F., M. R. Mahdavi, H. Jalali, B.
  • Talebi, P. Roshan, dan M. Mahdavi. 2014. Evaluation
  • of Gene Mutations Involved in Drug Resistance in
  • Mycobacterium tuberculosis Strains Derived from
  • Tuberculosis Patients in Mazandaran, Iran, 2013. Int J
  • Mol Cell Med Summer. Volume 3(3): 191-195.
  • [5] Silva, P. E. A. D., dan J. C. Palomino. 2011.
  • Molecular Basis and Mechanisms of Drug Resistance
  • in Mycobacterium tuberculosis: Classical and New
  • Drugs. Journal of Antimicrobial Chemotherapy.
  • Volume 66(7): 1417-1430.
  • [6] Tseng, S. T., C. H. Tai, C. R. Li, C. F. Lin, dan Z. Y.
  • Shi. 2015. The Mutations of katG and inhA Genes of
  • Isoniazid-Resistant Mycobacterium tuberculosis
  • Isolates in Taiwan. Journal of Microbiology,
  • Imunology and Infection. Volume 48: 249-255.
  • [7] Murray, J. L., P. Hu, dan D. A. Shafer. 2014. Seven
  • Novel Probe System for Real-Time PCR Provide
  • Absolute Single-Base Discrimination, Higher
  • Signaling, and Generic Components. The Journal of
  • Molecular Diagnostics. Vol. 16 (6): 627-638.
  • [8] Suryadi, T. 2013. Identifikasi Mutasi Gen katG Pada
  • Isolat P10 dan 151 Mycobacterium tuberculosis
  • Multidrug Resistance di Bali Dengan Metode
  • Polymerase Chain Reaction. Skripsi. Universitas
  • Udayana, Bukit Jimbaran: 1-39.
  • [9] Deniariasih, N. W. 2013. Deteksi Mutasi Pada Gen
  • katG (Fragmen 0,7 kb) Isolat 86 dan P11
  • Mycobacterium tuberculosis Multidrug Resistance
  • Dengan Teknik Polymerase Chain Reaction (PCR).
  • Skripsi. Universitas Udayana, Bukit Jimbaran: 1-39.
  • [10] Hazbon, M. H., M. Brimacombe, M. B. D.Valle, M.
  • Cavatore, M. I. Guerrero, M. V. Basil, H. B. Jacobe,
  • C. Lavender, J. Fyfe, L. G. Garcia, C. I. Leon, M.
  • Bose, F. Chaves, M. Murray, K. D. Eisenach, J. S.
  • Osornio, M. D. Cave, A. P. D. Leon, dan D. Alland.
    1. Population Genetics Study of Isoniazid
  • Resistance Mutations and Evolution of Multidrug-
  • Resistant Mycobacterium tuberculosis. Antimicrobial
  • Agents and Chemotherapy. Volume 50(8): 2640-2649.
  • [11] Bostanabad, S. Z., L. P. Titov, A. Bahrmand, S. A.
  • Nojoumi. 2008. Detection of Mutation in Isoniazid-
  • Resistant Mycobacterium tuberculosis Isolates From
  • Tuberculosis Patient in Belarus. Indian Journal of
  • Medical Microbiology. Volume 26(2): 143-147.
  • [12] Jagielski, T., Z. Bakuta, K. Roeske, M. Kaminski, A.
  • Napiorkowska, E. A. Kopec, Z. Zwolska, dan J.
  • Bielecki. 2014. Detection of Mutations Associated
  • With Isoniazid Resistance in Multidrug-Resistant
  • Mycobacterium tuberculosis Clinical Isolates. J.
  • Antimicrob Chemother. Volume 69(4): 2369-2375.
  • [13] Navarro, E., G. S. Heras, M. J. Castano, dan J. Solera.
    1. Real-Time PCR Detection Chemistry. Clinica
  • Chimica Acta. Volume 439: 231-250.
  • [14] Espy, M. J., J. R. Uhl, L. M. Sloan, S. P. Buckwalter,
  • M. F. Jones, E. A. Vetter, J. D. C. Yao, N. L.
  • Wengenack, dan J. E. Rosenblatt. 2006. Real-Time
  • PCR in Clinical Microbiology: Applications for
  • Routine Laboratory Testing. Journal of Clinical
  • Microbiology Review. Volume 19(1): 165-256.
  • [15] Dorak, M. T. 2006. Real-Time PCR. New York:
  • Taylor and Francis Group.1-30.
  • [16] McPherson, M., dan S. Moller. 2006. PCR Edisi 2.
  • New York: Taylor & Francis Group. 209-305.
  • [17] Walker, J.M. dan Rapley, R. 2008.Medical
  • Biomethods Handbook.Totowa, New Jersey: Humana
  • Press Inc. 13-341.
  • [18] Wada, T., S. Maeda, A. Tamaru, S. Imai, A. Hase, dan
  • K. Kobayashi.2004. Dual-Probe Assay For Rapid
  • Detection of Drug-Resistant Mycobacterium
  • tuberculosis by Real-Time PCR. Journal of Clinical
  • Microbiology. Volume 42(11): 5277-5285.
  • [19] Kurreck, J. dan C. A. Stein. 2015. Molecular
  • Medicine. Jerman: Wiley-VCH. 37-51.
  • [20] Chou, C.C., C. C. H. Chen, T. T. Lee, dan K. Peck.
    1. Optimization of Probe Length and The Number
  • of Probes Per Gene for Optimal Microarray Analysis
  • of Gene Expression. Nucleic Acid Research. Volume
  • 32(12): 99-107.
  • [21] Kalendar, R., D. Lee, dan A. H. Schulman. 2011. Java
  • Web Tools for PCR, In Silico PCR, and
  • Oligonucleotide Assembly and Analysis. Genomics.
  • Volume 98: 137-144.
  • [22] Yilmaz, L. S., S. Parnerkar, dan D. D. R. Noguera.
    1. mathFISH, A Web Tool That Uses
  • Thermodynamics-Based Mathematical Models for In
  • Silico Evaluation of Oligonucleotide Probes for
  • Fluorescence In Situ Hybridization. Applied And
  • Environmental Microbiology. Volume 77(3): 1118-
  • 1122.
  • [23] Zhang, Y. dan W. W. Yew. 2009. Mechanisms of
  • Drug Resistance in Mycobacterium tuberculosis. Int.
  • J. Tuberc Lung Dis. Volume 13(11): 1320-1330.
  • [24] Lina, M., B. Bela, dan A. Yasmon. 2009. Deteksi
  • Mutasi Gen katG Mycobacterium tuberculosis Dengan
  • Metode PCR (Polymerase Chain Reaction)-
  • Hibridisasi Dot Blot Menggunakan Pelacak
  • Oligonukleotida Bertanda 32P. Jurnal ilmiah Aplikasi
  • Isotop dan Radiasi. Volume 5 (1): 54-67.
  • [25] Smith, J. E. 2009. Biotechnology 5th Edition. New
  • York: Cambridge University Press. 41-44.
  • [26] Anonim a. 2004. Assay Formats for Use in Real-Time
  • PCR. Technical Note. Jerman: Roche Applied
  • Science. 1-13. Available at: https://lifescience.
  • roche.com/wcsstore/RASCatalogAssetStore/Articles/A
  • ssay%20Format%20for%20use%20in%20Real-
  • Time%20PCR.pdf. (Cited March 17, 2016).
  • [27] Alvandi, E. dan F Koohdani. 2014. Zip Nucleic Acid:
  • A New Reliable Method To Increase The Melting
  • Temperature of Real-Time PCR Probes. Journal of
  • Diabetes & Metabolic Disorders. Vol. 13 (26): 1-4.
  • [28] Livak, K. J., S. J. A. Flood, J. Marmaro, W. Giusti,
  • dan K. Deetz. 1995. Oligonucleotides With
  • Fluorescent Dyes At Opposite Ends Provide A
  • Quenched Probe System Useful For Detecting PCR
  • Product and Nucleic Acid Hybridization. Genome
  • Research. Vol. 4: 357-362.
  • [29] Anonim d. 2001. Allelic Discrimination Using The 5’
  • Nuclease Assay. USA: Applied Biosystems. 1-8.
  • http://www.austincc.edu/mlt/mdfund/mdfund
  • _Unit11AllelicDiscrimination.pdf. (Cited March 14,
  • 2016).
  • [30] Logan, J., K. Edwards, dan N. Saunders. 2009. Real-
  • Time PCR: Current Technology and Applications.
  • London: Caister Academic Press. 1-27. Available
  • at:www.horizonpress.com/realtimepcr.
  • http://citeseerx.
  • ist.psu.edu/viewdoc/download?doi=10.1.1.261.223&re
  • p=rep1&type=pdf. (Cited April 23, 2016).
  • [31] Anonim b. 2006. Real-Time PCR Applications Guide.
  • Bulletin 5279. USA: Bio-Rad Laboratories Inc. 1-100.
  • Available at: www.bio-rad.com/webroot/
  • web/pdf/lsr/literature/Bulletin_5279.pdf. (Cited
  • February 15, 2016).
  • [32] Holland, P. M., R. D. Abramcon, R. Watson. Dan D.
  • H. Gelfand. 1991. Detection of Spesific Polymerase
  • Chain Reaction Product By Utilizing The 5’→3’
  • Exonuclease Activity of Thermus aquaticus DNA
  • Polymerase. Proc Natl Acad Sci Biochemistry. Vol.
  • 88: 7276-7280.
  • [33] Liu, H., H. Wang, Z. Shi, H. Wang, C. Yang, S. Silke,
  • W. Tan, dan Z. Lu. 2006. Taqman Probe Array For
  • Quantitative Detection of DNA Targets. Nucleic Acids
  • Research. Vol. 34(1): 1-8.
  • [34] Handoyo, D., dan A. Rudiretna. 2000. Prinsip Umum
  • dan Pelaksanaan Polymerase Chain Reaction
  • (PCR).Unitas. Vol. 9 (1): 17-29.
  • [35] Patel, N. K., dan N. Prakash. 2013. Principle and
  • Tools For Primer Design. Atmiya Spandan Biological
  • Sciences. Vol. 1 (1): 79-95.
  • [36] Bishop, J. L., S. A. Campbell, P. Farrell, M.
  • Fitzgerald, M. Haugen, W. Kocmond, D.E. Madden,
  • W. E. Murray, dan D. H. Persing. 2015. Designing
  • Real-Time Assays on the SmartCycler® II System.
  • United State: Cepheid Technical Support. 1-
  • 8.Available at: http://www.cepheid.com/us/
  • component/phocadownload/category/2-
  • support?download=8:smart-note-6-1. (Cited April 20,
  • 2016).
  • [37] Proudnikov, D., V. Yuferov, Y. Zhou, K. S. Laforge,
  • A. Ho, M. J. Kreek. 2003. Optimizing Primer-Probe
  • Design for Fluorescent PCR. Journal of Neuroscience
  • Methods. Vol 123: 31-45.
  • [38] Lazaro, R. D. dan M. Hernandez. 2013. Real-Time
  • PCR in Food Science Current Technology and
  • Applications. Norfolk, UK: Caister Academic Press. 5-
  • 12.
  • [39] Mulle, J. G., V. C. Patel, S. T. Warren, M. R. Hegde,
  • D. J. Cutler, dan M. E. Zwick.2010. Empirical
  • Evaluation of Oligonucleotide Probe Selection for
  • DNA Microarrays. Plos One. Vol. 5 (3): 1-7.
  • [40] Anonim c. 2006. Beacon Designer 5.10 Manual.
  • Corona Way: USA: Premier Biosoft International. 22-
    1. Available at:
  • http://211.69.128.172/tlli/upfile/doc/7584c8_Beacon%
  • 20Designer%20510Manual.pdf. (Cited February 25,
  • 2016).
  • [41] Kubista, M., J. M. Andrade, M. Bengtsson, A.
  • Forootan, J. Jonak, K. Lind, R. Sindelka, R. Sjoback,
  • B. Sjogreen, L. Strombom, A. Stahlberg, dan N. Zoric.
    1. The Real-Time Polymerase Chain Reaction.
  • Molecular Aspects of Medicine Review. Vol. 27: 95-
  • 125.
  • [42] Yuwono, T. 2008. Biologi Molekuler. Jakarta:
  • Penerbit Erlangga. 49-74.
  • [43] Borah, P. 2011. Primer Designing For PCR.Sci Vis.
  • Vol. 3: 134-136.
  • [44] Rychlik, W. 2008.Oligo Primer Analysis Software
  • Version 7. USA: Molecular Biology Insights Inc. 90-
  • 123.
  • [45] Nazarenko, I., R. Pires, B. Lowe, M. Obaidy, dan A.
  • Rashtchian. 2002. Effect of Primary and Secondary
  • Structure of Oligodeoxyribonucleotides on The
  • Fluorescent Properties of Conjugated Dyes. Nucleic
  • Acids Research. Volume 30 (9): 2089-2195.
  • [46] Mackay, I. M. 2007. Real-Time PCR in Microbiology
  • From Diagnosis to Characterization. Norfolk, Uk:
  • Caister Academic Press. 1-64

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https://jurnal.harianregional.com/jhsm/full-36991

Published

2018-01-25

How To Cite

RAHMARYANI, I Gusti A. A. Santhi et al. DNA Probe Design for Detection Mutation at Codon 315 In katG Gene of Mycobacterium Tuberculosis to Real-Time Polymerase Chain Reaction.Journal of Health Sciences and Medicine, [S.l.], v. 1, n. 2, p. 31-41, jan. 2018. ISSN 2622-0555. Available at: https://ojs.unud.ac.id/index.php/JHSM/article/view/36991. Date accessed: 28 Aug. 2025. doi:https://doi.org/10.24843/JHSM.2017.v01.i02.p08.

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