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Insertion Element IS6110-Based Restriction Fragment Length Polymorphism Genotyping of Mycobacterium tuberculosis

  • Robin M. Warren
  • Paul D. van Helden
  • Nicolaas C. Gey van Pittius
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 465)

Abstract

DNA fingerprinting techniques are based on genome variation and form the basis of molecular epidemiology studies of tuberculosis. A number of markers are in use for the molecular differentiation of Mycobacterium tuberculosis isolates by DNA fingerprinting. One of these markers is the IS6110 insertion element, which may be present in up to 25 copies per M. tuberculosis genome. Variation in both the number and location of the IS6110 elements makes it a very useful marker of strain genotype. IS6110-based DNA fingerprinting is globally considered as the reference genotyping technique for M. tuberculosis isolates. This method is based on visualization of restriction fragment length polymorphisms using a labeled probe derived from IS6110. In this chapter, the method of IS6110 DNA fingerprinting is explained in such a way that it can be easily duplicated by molecular epidemiologists and will give reproducible results.

Keywords

culture DNA extraction ECL labeling IS6110 insertion element RFLP Southern hybridization transposition 

Notes

Acknowledgments

We would like to thank Ms. Talita Lotz for providing the IS6110-based RFLP image.

References

  1. 1.
    Hermans, P. W., van Soolingen, D., Bik, E. M., de Haas, P. E., Dale, J. W., and van Embden, J. D. (1991). Insertion element IS987 from Mycobacterium bovis BCG is located in a hot-spot integration region for insertion elements in Mycobacterium tuberculosis complex strains. Infect. Immun. 59, 2695–2705.PubMedGoogle Scholar
  2. 2.
    Groenen, P. M., Bunschoten, A. E., van Soolingen, D., and van Embden, J. D. (1993). Nature of DNA polymorphism in the direct repeat cluster of Mycobacterium tuberculosis; application for strain differentiation by a novel typing method. Mol. Microbiol. 10, 1057–1065.PubMedCrossRefGoogle Scholar
  3. 3.
    Ross, B. C., Raios, K., Jackson, K., and Dwyer, B. (1992). Molecular cloning of a highly repeated DNA element from Mycobacterium tuberculosis and its use as an epidemiological tool. J. Clin. Microbiol. 30, 942–946.PubMedGoogle Scholar
  4. 4.
    Supply, P., Mazars, E., Lesjean, S., Vincent, V., Gicquel, B., and Locht, C. (2000). Variable human minisatellite-like regions in the Mycobacterium tuberculosis genome. Mol. Microbiol. 36, 762–771.PubMedCrossRefGoogle Scholar
  5. 5.
    Thierry, D., Brisson-Noel, A., Vincent-Levy-Frebault, V., Nguyen, S., Guesdon, J. L., and Gicquel, B. (1990). Characterization of a Mycobacterium tuberculosis insertion sequence, IS6110, and its application in diagnosis. J. Clin. Microbiol. 28, 2668–2673.PubMedGoogle Scholar
  6. 6.
    Kremer, K., van Soolingen, D., Putova, I., and Kubin, M. (1996). Use of IS6110 DNA fingerprinting in tracing man-to-man transmission of Mycobacterium tuberculosis in the Czech Republic. Cent. Eur. J. Public Health 4, 3–6.PubMedCrossRefGoogle Scholar
  7. 7.
    van Embden, J. D., Cave, M. D., Crawford, J. T., Dale, J. W., Eisenach, K. D., Gicquel, B. et al. (1993). Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. J. Clin. Microbiol. 31, 406–409.PubMedGoogle Scholar
  8. 8.
    Dale, J. W. (1995). Mobile genetic elements in mycobacteria. Eur. Respir. J. Suppl 20, 633s–648s.Google Scholar
  9. 9.
    Alland, D., Kalkut, G. E., Moss, A. R., McAdam, R. A., Hahn, J. A., Bosworth, W. et al. (1994). Transmission of tuberculosis in New York City. An analysis by DNA fingerprinting and conventional epidemiologic methods. N. Engl. J. Med. 330, 1710–1716.PubMedCrossRefGoogle Scholar
  10. 10.
    Small, P. M., Hopewell, P. C., Singh, S. P., Paz, A., Parsonnet, J., Ruston, D. C. et al. (1994). The epidemiology of tuberculosis in San Francisco. A population-based study using conventional and molecular methods. N. Engl. J. Med. 330, 1703–1709.PubMedCrossRefGoogle Scholar
  11. 11.
    Warren, R., Hauman, J., Beyers, N., Richardson, M., Schaaf, H. S., Donald, P. et al. (1996). Unexpectedly high strain diversity of Mycobacterium tuberculosis in a high-incidence community. S. Afr. Med. J. 86, 45–49.PubMedGoogle Scholar
  12. 12.
    Hermans, P. W., Messadi, F., Guebrexabher, H., van Soolingen, D., de Haas, P. E., Heersma, H. et al. (1995). Analysis of the population structure of Mycobacterium tuberculosis in Ethiopia, Tunisia, and The Netherlands: usefulness of DNA typing for global tuberculosis epidemiology. J. Infect. Dis. 171, 1504–1513.PubMedCrossRefGoogle Scholar
  13. 13.
    van Rie, A., Warren, R., Richardson, M., Victor, T. C., Gie, R. P., Enarson, D. A. et al. (1999). Exogenous reinfection as a cause of recurrent tuberculosis after curative treatment. N. Engl. J. Med. 341, 1174–1179.PubMedCrossRefGoogle Scholar
  14. 14.
    van Rie, A., Warren, R. M., Beyers, N., Gie, R. P., Classen, C. N., Richardson, M. et al. (1999). Transmission of a multidrug-resistant Mycobacterium tuberculosis strain resembling “strain W” among noninstitutionalized, human immunodeficiency virus-seronegative patients. J. Infect. Dis. 180, 1608–1615.PubMedCrossRefGoogle Scholar
  15. 15.
    van Rie, A., Warren, R., Richardson, M., Gie, R. P., Enarson, D. A., Beyers, N. et al. (2000). Classification of drug-resistant tuberculosis in an epidemic area. Lancet 356, 22–25.PubMedCrossRefGoogle Scholar
  16. 16.
    Michalak, K., Austin, C., Diesel, S., Bacon, M. J., Zimmerman, P., and Maslow, J. N. (1998). Mycobacterium tuberculosis infection as a zoonotic disease: transmission between humans and elephants. Emerg. Infect. Dis. 4, 283–287.PubMedCrossRefGoogle Scholar
  17. 17.
    Verver, S., Warren, R. M., Munch, Z., Richardson, M., van der Spuy, G. D., Borgdorff, M. W. et al. (2004). Proportion of tuberculosis transmission that takes place in households in a high-incidence area. Lancet 363, 212–214.PubMedCrossRefGoogle Scholar
  18. 18.
    Heyns, L., Gie, R. P., Goussard, P., Beyers, N., Warren, R. M., and Marais, B. J. (2006). Nosocomial transmission of Mycobacterium tuberculosis in kangaroo mother care units: a risk in tuberculosis-endemic areas. Acta Paediatr. 95, 535–539.PubMedCrossRefGoogle Scholar
  19. 19.
    Richardson, M., Carroll, N. M., Engelke, E., van der Spuy, G. D., Salker, F., Munch, Z. et al. (2002). Multiple Mycobacterium tuberculosis strains in early cultures from patients in a high-incidence community setting. J. Clin. Microbiol. 40, 2750–2754.PubMedCrossRefGoogle Scholar
  20. 20.
    Small, P. M., McClenny, N. B., Singh, S. P., Schoolnik, G. K., Tompkins, L. S., and Mickelsen, P. A. (1993). Molecular strain typing of Mycobacterium tuberculosis to confirm cross-contamination in the mycobacteriology laboratory and modification of procedures to minimize occurrence of false-positive cultures. J. Clin. Microbiol. 31, 1677–1682.PubMedGoogle Scholar
  21. 21.
    Warren, R., de Kock, M., Engelke, E., Myburgh, R., Gey, v. P., Victor, T. et al. (2006). Safe Mycobacterium tuberculosis DNA extraction method that does not compromise integrity. J. Clin. Microbiol. 44, 254–256.PubMedCrossRefGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Robin M. Warren
    • 1
  • Paul D. van Helden
    • 2
  • Nicolaas C. Gey van Pittius
    • 2
  1. 1.DST/NRF Centre of Excellence in Biomedical Tuberculosis Research / MRC Centre for Molecular and Cellular Biology, Division of Molecular Biology and Human Genetics, Department of Biomedical Sciences, Faculty of Health SciencesStellenbosch UniversityTygerbergSouth Africa
  2. 2.Centre for Infectious Disease, Institute of Cell and Molecular ScienceBarts and The London, Queen Mary’s School of Medicine and DentistryLondonUK

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