| Peer-Reviewed

The Reproducibility of the Multiplex RAPD-PCR Assay in Genotyping of Mycobacterium Tuberculosis Isolates from Sulawesi, Indonesia

Received: 20 February 2014     Published: 20 March 2014
Views:       Downloads:
Abstract

Background: Random Amplified Polymorphic DNA (RAPD) assay has recently emerged as a genotyping method which is both robust and highly discriminatory for bacterial strain differentiation. However, RAPD assessment for Mycobacterium tuberculosiscomplex (MTC) isolates is still limited. Despite its simplicity and rapidity, conventional RAPD also has low reproducibility due to its sensitivity to several factors. Therefore we studied the feasibility of an RAPD-PCR assay to define the genetic diversity of MTC isolates and to evaluate its reproducibility.Methods:493clinical MTC isolates from the island of Sulawesi in Eastern Indonesia, collected from 2005-2012were subjected to Multiplex RAPD assay using 11 random decamer primers instead of one primer which is common in conventional RAPD. All 11 primers were found to be differentiated and produced specific RAPD profiles. The polymorphic amplicons served as RAPD markers for MTC. The dendrograms, obtained by different primers, showed the discriminatory ability of the primers.Results:Multiplex RAPD-PCR results show that the majority of the isolates from South Sulawesi, Southeast Sulawesi andCentral Sulawesiin eastern region of Indonesia belong to group MT-C (80.7%, 80.0% and 62.6 % respectively) with result reproducibility as high as 100%. Conclusion:Molecular typing with multiplex RAPD-PCR is a powerful approach to show the genetic heterogeneity of MTC isolates. The discrimination power and reproducibility of this multiple loci-based RAPD was higher than conventional fewer loci-targeted RAPD.

Published in American Journal of Clinical and Experimental Medicine (Volume 2, Issue 2)
DOI 10.11648/j.ajcem.20140202.12
Page(s) 14-21
Creative Commons

This is an Open Access article, distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution and reproduction in any medium or format, provided the original work is properly cited.

Copyright

Copyright © The Author(s), 2014. Published by Science Publishing Group

Keywords

RAPD-PCR, Genotyping, Mycobacterium Tuberculosis Complex, Sulawesi Indonesia

References
[1] WHO: Global Tuberculosis Report 2013. WHO Press 2013:http://www.who.int/tb/publications/global_report/en/.
[2] Connell DW, Berry M, Cooke G, Kon OM: Update on tuberculosis: TB in the early 21st century. European respiratory review : an official journal of the European Respiratory Society 2011, 20(120):71-84.
[3] Jain SK, Lamichhane G, Nimmagadda S, Pomper MG, Bishai WR: Antibiotic treatment of tuberculosis: old problem, new solution. Microbes Infect 2008, 3:6.
[4] NhlemaSimwaka B, Benson T, Salaniponi FM, Theobald SJ, Squire SB, Kemp JR: Developing a socio-economic measure to monitor access to tuberculosis services in urban Lilongwe, Malawi. Int J Tuberc Lung Dis 2007, 11(1):65-71.
[5] Caminero JA: Multidrug-resistant tuberculosis: epidemiology, risk factors and case finding. Int J Tuberc Lung Dis 2010, 14(4):382-390.
[6] Golub JE, Mohan CI, Comstock GW, Chaisson RE: Active case finding of tuberculosis: historical perspective and future prospects. Int J Tuberc Lung Dis 2005, 9(11):1183-1203.
[7] Jassal MS, Bishai WR: Epidemiology and challenges to the elimination of global tuberculosis. Clin Infect Dis 2010, 50 Suppl 3:S156-164.
[8] Kaufmann SH, Schaible UE: 100th anniversary of Robert Koch's Nobel Prize for the discovery of the tubercle bacillus. Trends Microbiol 2005, 13(10):469-475.
[9] Hatta M, Sultan AR, Tandirogang N, Masjudi, Yadi: Detection and identification of mycobacteria in sputum from suspected tuberculosis patients. BMC Res Notes 2010, 3:72.
[10] Takashima T, Iwamoto T: [New era in molecular epidemiology of tuberculosis in Japan]. Kekkaku: [Tuberculosis] 2006, 81(11):693-707.
[11] Gibson A, Brown T, Baker L, Drobniewski F: Can 15-locus mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis provide insight into the evolution of Mycobacterium tuberculosis? Appl Environ Microbiol 2005, 71(12):8207-8213.
[12] Blackwood KS, Al-Azem A, Elliott LJ, Hershfield ES, Kabani AM: Conventional and molecular epidemiology of tuberculosis in Manitoba. BMC infectious diseases 2003, 3:18.
[13] Scott AN, Menzies D, Tannenbaum TN, Thibert L, Kozak R, Joseph L, Schwartzman K, Behr MA: Sensitivities and specificities of spoligotyping and mycobacterial interspersed repetitive unit-variable-number tandem repeat typing methods for studying molecular epidemiology of tuberculosis. J ClinMicrobiol 2005, 43(1):89-94.
[14] Mostowy S, Behr MA: The origin and evolution of Mycobacterium tuberculosis. Clinics in chest medicine 2005, 26(2):207-216, v-vi.
[15] Korzekwa K, Polok K, Zielinski R: Application of DNA markers to estimate genetic diversity of Mycobacterium tuberculosis strains. Polish journal of microbiology / PolskieTowarzystwoMikrobiologow = The Polish Society of Microbiologists 2006, 55(1):19-24.
[16] Tazi L, El Baghdadi J, Lesjean S, Locht C, Supply P, Tibayrenc M, Banuls AL: Genetic diversity and population structure of Mycobacterium tuberculosis in Casablanca, a Moroccan city with high incidence of tuberculosis. J ClinMicrobiol 2004, 42(1):461-466.
[17] Maidin MA, Lidjaja A, Hatta M: Random Amplified Polymorphic DNA Analysis of Mycobacterium tuberculosis isolates Resistant to Isoniazid in Indonesia. J Tuberculosis Res 2013, 1:10-13.
[18] Singh JP, Verma R, Chaudhuri P: Random amplified polymorphic DNA (RAPD) analysis of Mycobacterium tuberculosis strains in India. Journal of veterinary science 2006, 7(2):181-187.
[19] deSoldenhoff R, Hatta M, Siro TW: Choosing the decolourizer and its strength to stain Mycobacterium leprae. Does it actually matter? Leprosy review 1998, 69(2):128-133.
[20] Hatta M, Eka W, Zaraswati D, Rosana A, Sabir M, Yadi., Mashyudi.: Effect decontamination in identification Mycobacterium tuberculosis by ZN staining and PCR technique (in Indonesia). YARSI Med J 2004, 12:17-24.
[21] Kent PT, Kubica GP: Public health mycobacteriology: A guide for level III laboratory. US Department of Health and Human Services, Center for Diseases Control, Atlanta 1985, 184.
[22] Hatta M, Smits HL: Detection of Salmonella typhi by nested polymerase chain reaction in blood, urine, and stool samples. The American journal of tropical medicine and hygiene 2007, 76(1):139-143.
Cite This Article
  • APA Style

    Mochammad Hatta, AndiRofian Sultan, Ressy Dwiyanti, Muhammad Sabir, Andini Febrianty, et al. (2014). The Reproducibility of the Multiplex RAPD-PCR Assay in Genotyping of Mycobacterium Tuberculosis Isolates from Sulawesi, Indonesia. American Journal of Clinical and Experimental Medicine, 2(2), 14-21. https://doi.org/10.11648/j.ajcem.20140202.12

    Copy | Download

    ACS Style

    Mochammad Hatta; AndiRofian Sultan; Ressy Dwiyanti; Muhammad Sabir; Andini Febrianty, et al. The Reproducibility of the Multiplex RAPD-PCR Assay in Genotyping of Mycobacterium Tuberculosis Isolates from Sulawesi, Indonesia. Am. J. Clin. Exp. Med. 2014, 2(2), 14-21. doi: 10.11648/j.ajcem.20140202.12

    Copy | Download

    AMA Style

    Mochammad Hatta, AndiRofian Sultan, Ressy Dwiyanti, Muhammad Sabir, Andini Febrianty, et al. The Reproducibility of the Multiplex RAPD-PCR Assay in Genotyping of Mycobacterium Tuberculosis Isolates from Sulawesi, Indonesia. Am J Clin Exp Med. 2014;2(2):14-21. doi: 10.11648/j.ajcem.20140202.12

    Copy | Download

  • @article{10.11648/j.ajcem.20140202.12,
      author = {Mochammad Hatta and AndiRofian Sultan and Ressy Dwiyanti and Muhammad Sabir and Andini Febrianty and Ahmad Adhyka and Nur Indah Purnamasari and Muhammad Reza Primaguna and Juhri Saning and Yusriani Mangarengi and Munawir Muhammad and Nataniel Tandirogang and Yadi Yasir and Masyhudi Amir},
      title = {The Reproducibility of the Multiplex RAPD-PCR Assay in Genotyping of Mycobacterium Tuberculosis Isolates from Sulawesi, Indonesia},
      journal = {American Journal of Clinical and Experimental Medicine},
      volume = {2},
      number = {2},
      pages = {14-21},
      doi = {10.11648/j.ajcem.20140202.12},
      url = {https://doi.org/10.11648/j.ajcem.20140202.12},
      eprint = {https://article.sciencepublishinggroup.com/pdf/10.11648.j.ajcem.20140202.12},
      abstract = {Background: Random Amplified Polymorphic DNA (RAPD) assay has recently emerged as a genotyping method which is both robust and highly discriminatory for bacterial strain differentiation. However, RAPD assessment for Mycobacterium tuberculosiscomplex (MTC) isolates is still limited. Despite its simplicity and rapidity, conventional RAPD also has low reproducibility due to its sensitivity to several factors. Therefore we studied the feasibility of an RAPD-PCR assay to define the genetic diversity of MTC isolates and to evaluate its reproducibility.Methods:493clinical MTC isolates from the island of Sulawesi in Eastern Indonesia, collected from 2005-2012were subjected to Multiplex RAPD assay using 11 random decamer primers instead of one primer which is common in conventional RAPD. All 11 primers were found to be differentiated and produced specific RAPD profiles. The polymorphic amplicons served as RAPD markers for MTC. The dendrograms, obtained by different primers, showed the discriminatory ability of the primers.Results:Multiplex RAPD-PCR results show that the majority of the isolates from South Sulawesi, Southeast Sulawesi andCentral Sulawesiin eastern region of Indonesia belong to group MT-C (80.7%, 80.0% and 62.6 % respectively) with result reproducibility as high as 100%. Conclusion:Molecular typing with multiplex RAPD-PCR is a powerful approach to show the genetic heterogeneity of MTC isolates. The discrimination power and reproducibility of this multiple loci-based RAPD was higher than conventional fewer loci-targeted RAPD.},
     year = {2014}
    }
    

    Copy | Download

  • TY  - JOUR
    T1  - The Reproducibility of the Multiplex RAPD-PCR Assay in Genotyping of Mycobacterium Tuberculosis Isolates from Sulawesi, Indonesia
    AU  - Mochammad Hatta
    AU  - AndiRofian Sultan
    AU  - Ressy Dwiyanti
    AU  - Muhammad Sabir
    AU  - Andini Febrianty
    AU  - Ahmad Adhyka
    AU  - Nur Indah Purnamasari
    AU  - Muhammad Reza Primaguna
    AU  - Juhri Saning
    AU  - Yusriani Mangarengi
    AU  - Munawir Muhammad
    AU  - Nataniel Tandirogang
    AU  - Yadi Yasir
    AU  - Masyhudi Amir
    Y1  - 2014/03/20
    PY  - 2014
    N1  - https://doi.org/10.11648/j.ajcem.20140202.12
    DO  - 10.11648/j.ajcem.20140202.12
    T2  - American Journal of Clinical and Experimental Medicine
    JF  - American Journal of Clinical and Experimental Medicine
    JO  - American Journal of Clinical and Experimental Medicine
    SP  - 14
    EP  - 21
    PB  - Science Publishing Group
    SN  - 2330-8133
    UR  - https://doi.org/10.11648/j.ajcem.20140202.12
    AB  - Background: Random Amplified Polymorphic DNA (RAPD) assay has recently emerged as a genotyping method which is both robust and highly discriminatory for bacterial strain differentiation. However, RAPD assessment for Mycobacterium tuberculosiscomplex (MTC) isolates is still limited. Despite its simplicity and rapidity, conventional RAPD also has low reproducibility due to its sensitivity to several factors. Therefore we studied the feasibility of an RAPD-PCR assay to define the genetic diversity of MTC isolates and to evaluate its reproducibility.Methods:493clinical MTC isolates from the island of Sulawesi in Eastern Indonesia, collected from 2005-2012were subjected to Multiplex RAPD assay using 11 random decamer primers instead of one primer which is common in conventional RAPD. All 11 primers were found to be differentiated and produced specific RAPD profiles. The polymorphic amplicons served as RAPD markers for MTC. The dendrograms, obtained by different primers, showed the discriminatory ability of the primers.Results:Multiplex RAPD-PCR results show that the majority of the isolates from South Sulawesi, Southeast Sulawesi andCentral Sulawesiin eastern region of Indonesia belong to group MT-C (80.7%, 80.0% and 62.6 % respectively) with result reproducibility as high as 100%. Conclusion:Molecular typing with multiplex RAPD-PCR is a powerful approach to show the genetic heterogeneity of MTC isolates. The discrimination power and reproducibility of this multiple loci-based RAPD was higher than conventional fewer loci-targeted RAPD.
    VL  - 2
    IS  - 2
    ER  - 

    Copy | Download

Author Information
  • Molecular Biology and Immunology Laboratory for Infectious Diseases, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia

  • Molecular Biology and Immunology Laboratory for Infectious Diseases, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia

  • Molecular Biology and Immunology Laboratory for Infectious Diseases, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia

  • Molecular Biology and Immunology Laboratory for Infectious Diseases, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia

  • Molecular Biology and Immunology Laboratory for Infectious Diseases, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia

  • Molecular Biology and Immunology Laboratory for Infectious Diseases, Faculty of Medicine, Hasanuddin University, Makassar, Indonesia

  • Department of Microbiology, Faculty of Medicine, Haluoleo University, Kendari, Indonesia

  • Department of Microbiology, Faculty of Medicine, Haluoleo University, Kendari, Indonesia

  • Department of Microbiology, Faculty of Medicine, Tadulako University, Palu, Indonesia

  • Department of Microbiology, Faculty of Medicine, Tadulako University, Palu, Indonesia

  • Department of Microbiology, Faculty of Medicine, Tadulako University, Palu, Indonesia

  • Department Microbiology, Faculty of Medicine, Mulawarman University, Samarinda, Indonesia

  • Department Microbiology, Faculty of Medicine, Mulawarman University, Samarinda, Indonesia

  • Department Microbiology, Faculty of Medicine, Mulawarman University, Samarinda, Indonesia

  • Sections