Typing Mycobacterium tuberculosis Using Variable Number Tandem Repeat Analysis

  • T.J. BrownEmail author
  • V.N. Nikolayevskyy
  • F.A. Drobniewski
Part of the Methods in Molecular Biology book series (MIMB, volume 465)


DNA-based typing has contributed to the understanding of M. tuberculosis epidemiology and evolution. IS6110 RFLP was the first method described and has been used in many epidemiologic investigations. Technological difficulties have hampered the widespread establishment of this method, and it has been found to be of little use in evolutionary studies. PCR-based methods such as spoligotyping and variable number tandem repeat (VNTR) analysis largely overcome these difficulties. Spoligotyping alone is of limited value in epidemiologic investigations due to low discrimination but can be useful in evolutionary studies. Panels of VNTR loci selected from the 59 polymorphic VNTRs described to date have been shown to be useful in both epidemiologic and evolutionary studies. A VNTR type is identified by, first, amplifying a series of PCR fragments each encompassing a different VNTR locus and, second, determining the PCR fragment sizes from which the number of repeats present is calculated. The repeat number present at a series of loci is used as numerical code to describe a type. This chapter describes a high-throughput automated method for VNTR analysis at 15 loci using a capillary fragment analyzer and a manual method using agarose gel analysis.


epidemiology ETR fragment analysis MIRU Mycobacterium tuberculosis typing VNTR 



We would like to thank Philip Supply for his advice and stimulating discussions.


  1. 1.
    van Soolingen, D., Hermans, P. W., de Haas, P. E., Soll, D. R. & van Embden, J. D. (1991). Occurrence and stability of insertion sequences in Mycobacterium tuberculosis complex strains: evaluation of an insertion sequence-dependent DNA polymorphism as a tool in the epidemiology of tuberculosis. J Clin Microbiol 29, 2578–2586.PubMedGoogle Scholar
  2. 2.
    Kamerbeek, J., Schouls, L., Kolk, A., van Agterveld, M., van Soolingen, D., Kuijper, S., Bunschoten, A., Molhuizen, H., Shaw, R., Goyal, M. & van Embden, J. (1997). Simultaneous detection and strain differentiation of Mycobacterium tuberculosis for diagnosis and epidemiology. J Clin Microbiol 35, 907–914.PubMedGoogle Scholar
  3. 3.
    Brudey, K., Driscoll, J., Rigouts, L., Prodinger, W., Gori, A., Al-Hajoj, S., Allix, C., Aristimuno, L., Arora, J., Baumanis, V., Binder, L., Cafrune, P., Cataldi, A., Cheong, S., Diel, R., Ellermeier, C., Evans, J., Fauville-Dufaux, M., Ferdinand, S., de Viedma, D., Garzelli, C., Gazzola, L., Gomes, H., Guttierez, M. C., Hawkey, P., van Helden, P., Kadival, G., Kreiswirth, B., Kremer, K., Kubin, M., Kulkarni, S., Liens, B., Lillebaek, T., Ly, H., Martin, C., Martin, C., Mokrousov, I., Narvskaia, O., Ngeow, Y., Naumann, L., Niemann, S., Parwati, I., Rahim, Z., Rasolofo-Razanamparany, V., Rasolonavalona, T., Rossetti, M. L., Rusch-Gerdes, S., Sajduda, A., Samper, S., Shemyakin, I., Singh, U., Somoskovi, A., Skuce, R., van Soolingen, D., Streicher, E., Suffys, P., Tortoli, E., Tracevska, T., Vincent, V., Victor, T., Warren, R., Yap, S., Zaman, K., Portaels, F., Rastogi, N. & Sola, C. (2006). Mycobacterium tuberculosis complex genetic diversity: mining the fourth international spoligotyping database (SpolDB4) for classification, population genetics and epidemiology. BMC Microbiol 6, 23.PubMedCrossRefGoogle Scholar
  4. 4.
    Kremer, K., van Soolingen, D., Frothingham, R., Haas, W. H., Hermans, P. W., Martin, C., Palittapongarnpim, P., Plikaytis, B. B., Riley, L. W., Yakrus, M. A., Musser, J. M. & van Embden, J. D. (1999). Comparison of methods based on different molecular epidemiological markers for typing of Mycobacterium tuberculosis complex strains: interlaboratory study of discriminatory power and reproducibility. J Clin Microbiol 37, 2607–2618.PubMedGoogle Scholar
  5. 5.
    Jeffreys, A. J., Wilson, V. & Thein, S. L. (1985). Individual-specific ‘fingerprints’ of human DNA. Nature 316, 76–79.PubMedCrossRefGoogle Scholar
  6. 6.
    Frothingham, R. & Meeker-O'Connell, W. (1998). Genetic diversity in the Mycobacterium tuberculosis complex based on variable numbers of tandem DNA repeats. Microbiology 144, 1189–1196.PubMedCrossRefGoogle Scholar
  7. 7.
    Supply, P., Mazars, E., Lesjean, S., Vincent, V., Gicquel, B. & Locht, C. (2000). Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol Microbiol 36, 762–771.PubMedCrossRefGoogle Scholar
  8. 8.
    Mazars, E., Lesjean, S., Banuls, A. L., Gilbert, M., Vincent, V., Gicquel, B., Tibayrenc, M., Locht, C. & Supply, P. (2001). High-resolution minisatellite-based typing as a portable approach to global analysis of Mycobacterium tuberculosis molecular epidemiology. Proc Natl Acad Sci U S A 98, 1901–1906.PubMedCrossRefGoogle Scholar
  9. 9.
    Hawkey, P. M., Smith, E. G., Evans, J. T., Monk, P., Bryan, G., Mohamed, H. H., Bardhan, M. & Pugh, R. N. (2003). Mycobacterial interspersed repetitive unit typing of Mycobacterium tuberculosis compared to IS6110-based restriction fragment length polymorphism analysis for investigation of apparently clustered cases of tuberculosis. J Clin Microbiol 41, 3514–3520.PubMedCrossRefGoogle Scholar
  10. 10.
    Le Fleche, P., Fabre, M., Denoeud, F., Koeck, J.-L. & Vergnaud, G. (2002). High resolution, on-line identification of strains from the Mycobacterium tuberculosis complex based on tandem repeat typing. BMC Microbiol 2, 37.PubMedCrossRefGoogle Scholar
  11. 11.
    Namwat, W., Luangsuk, P. & Palittapongarnpim, P. (1998). The genetic diversity ofMycobacterium tuberculosis strains in Thailand studied by amplification of DNA segments containing direct repetitive sequences. Int J Tuberculosis Lung Dis 2, 153–159.Google Scholar
  12. 12.
    Roring, S., Scott, A., Brittain, D., Walker, I., Hewinson, G., Neill, S. & Skuce, R. (2002). Development of variable-number tandem repeat typing of Mycobacterium bovis: comparison of results with those obtained by using existing exact tandem repeats and spoligotyping. J Clin Microbiol 40, 2126–2133.PubMedCrossRefGoogle Scholar
  13. 13.
    Skuce, R. A., McCorry, T. P., McCarroll, J. F., Roring, S. M. M., Scott, A. N., Brittain, D., Hughes, S. L., Hewinson, R. G. & Neill, S. D. (2002). Discrimination of Mycobacterium tuberculosis complex bacteria using novel VNTR-PCR targets. Microbiology 148, 519–528.PubMedGoogle Scholar
  14. 14.
    Smittipat, N., Billamas, P., Palittapongarnpim, M., Thong-On, A., Temu, M. M., Thanakijcharoen, P., Karnkawinpong, O. & Palittapongarnpim, P. (2005). Polymorphism of variable-number tandem repeats at multiple loci in Mycobacterium tuberculosis. J Clin Microbiol 43, 5034–5043.PubMedCrossRefGoogle Scholar
  15. 15.
    Spurgiesz, R. S., Quitugua, T. N., Smith, K. L., Schupp, J., Palmer, E. G., Cox, R. A. & Keim, P. (2003). Molecular typing of mycobacterium tuberculosis by using nine novel variable-number tandem repeats across the Beijing family and low-copy-number IS6110 isolates. J Clin Microbiol 41, 4224–4230.PubMedCrossRefGoogle Scholar
  16. 16.
    van Deutekom, H., Supply, P., de Haas, P. E. W., Willery, E., Hoijng, S. P., Locht, C., Coutinho, R. A. & van Soolingen, D. (2005). Molecular typing of Mycobacterium tuberculosis by mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis, a more accurate method for identifying epidemiological links between patients with tuberculosis. J Clin Microbiol 43, 4473–4479.PubMedCrossRefGoogle Scholar
  17. 17.
    Gopaul, K. K., Brown, T. J., Gibson, A. L., Yates, M. D. & Drobniewski, F. A. (2006). Progression toward an improved DNA amplification-based typing technique in the study of Mycobacterium tuberculosis epidemiology. J Clin Microbiol 44, 2492–2498.PubMedCrossRefGoogle Scholar
  18. 18.
    Gibson, A., Brown, T., Baker, L. & Drobniewski, F. (2005). Can 15-locus mycobacterial interspersed repetitive unit-variable-number tandem repeat analysis provide insight into the evolution of Mycobacterium tuberculosis? Appl Environ Microbiol 71, 8207–8213.PubMedCrossRefGoogle Scholar
  19. 19.
    Evans, J. T., Hawkey, P. M., Smith, E. G., Boese, K. A., Warren, R. E. & Hong, G. (2004). Automated high-throughput mycobacterial interspersed repetitive unit typing of Mycobacterium tuberculosis strains by a combination of PCR and nondenaturing high-performance liquid chromatography. J Clin Microbiol 42, 4175–4180.PubMedCrossRefGoogle Scholar
  20. 20.
    Supply, P., Lesjean, S., Savine, E., Kremer, K., van Soolingen, D. & Locht, C. (2001). Automated high-throughput genotyping for study of global epidemiology of Mycobacterium tuberculosis based on mycobacterial interspersed repetitive units. J Clin Microbiol 39, 3563–3571.PubMedCrossRefGoogle Scholar
  21. 21.
    Kwara, A., Schiro, R., Cowan, L. S., Hyslop, N. E., Wiser, M. F., Roahen Harrison, S., Kissinger, P., Diem, L. & Crawford, J. T. (2003). Evaluation of the epidemiologic utility of secondary typing methods for differentiation of Mycobacterium tuberculosis isolates. J Clin Microbiol 41, 2683–2685.PubMedCrossRefGoogle Scholar
  22. 22.
    Supply, P., Allix, C., Lesjean, S., Cardoso-Oelemann, M., Rusch-Gerdes, S., Willery, E., Savine, E., de Haas, P., van Deutekom, H., Roring, S., Bifani, P., Kurepina, N., Kreiswirth, B., Sola, C., Rastogi, N., Vatin, V., Gutierrez, M. C., Fauville, M., Niemann, S., Skuce, R., Kremer, K., Locht, C. & van Soolingen, D. (2006). Proposal for standardization of optimized mycobacterial interspersed repetitive unit-variable number tandem repeat typing of Mycobacterium tuberculosis. J Clin Microbiol 44, 4498–4510.PubMedCrossRefGoogle Scholar
  23. 23.
    Nikolayevskyy, V., Gopaul, K., Balabanova, Y., Brown, T., Fedorin, I. & Drobniewski, F. (2006). Differentiation of tuberculosis strains in a population with mainly Beijing-family strains. Emerg Infect Dis 12, 1406–1413.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press, a part of Springer Science+Business Media, LLC 2009

Authors and Affiliations

  • T.J. Brown
    • 1
    Email author
  • V.N. Nikolayevskyy
    • 2
  • F.A. Drobniewski
    • 2
  1. 1.HPA MRU, Queen Mary’s School of Medicine and DentistryLondonUK
  2. 2.Health Protection Agency Mycobacterium Reference UnitBarts and The London, Queen Mary’s School of Medicine and DentistryLondonUK

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