Skip to main content

Genotyping of Mycobacterium tuberculosis Clinical Isolates Using IS6110-Based Restriction Fragment Length Polymorphism Analysis

  • Protocol
  • First Online:
Molecular Epidemiology of Microorganisms

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 551))

Abstract

A number of phylogenetic studies of Mycobacterium tuberculosis have suggested a highly clonal population structure. Despite the extreme homogeneity of M. tuberculosis strains, the genome is punctuated by a number of polymorphic regions that give rise to sufficient diversity, thus forming the basis for molecular epidemiologic studies of tuberculosis. As such, insertion sequence (IS) 6110, which is unique to members of the M. tuberculosis complex and is present in variable numbers and in discrete genomic locales among strains, has been extensively used in molecular epidemiologic studies. Genotyping, using IS6110-based restriction fragment length polymorphism (RFLP), was standardized by the international community, and this has facilitated inter- and intralaboratory comparison, thereby serving as a model system for subspeciation of M. tuberculosis. When IS6110-based RFLP was used in conjunction with conventional epidemiologic data, its utility was realized. In this chapter, we discuss the basic methodology for conducting IS6110-based RFLP and analyzing the resulting hybridization profiles.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 84.99
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.00
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 109.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. World Health Organization (2007). Tuberculosis Facts. Available at: http://www.who.int/tb/publications/2007/factsheet_2007.pdf.

    Google Scholar 

  2. Zignol, M., Hosseini, M. S., Wright, A., Weezenbeek, C. L., Nunn, P., Watt, C. J., et al (2006). Global incidence of multidrug-resistant tuberculosis. J. Infect. Dis. 194, 479– 485.

    Article  Google Scholar 

  3. Mahillon, J., and Chandler, M. (1998). Insertion sequences. Microbiol. Mol. Biol. Rev. 62, 725–774.

    CAS  Google Scholar 

  4. McAdam, R. A., Hermans, P. W., van Soolingen, D., Zainuddin, Z. F., Catty, D., van Embden, J. D., et al (1990). Characterization of a Mycobacterium tuberculosis insertion sequence belonging to the IS3 family. Mol. Microbiol. 4, 1607–1613.

    Article  PubMed  CAS  Google Scholar 

  5. McEvoy, C. R., Falmer, A. A., Gey van Pittius, N. C., Victor, T. C., van Helden, P. D., and Warren, R. M. (2007). The role of IS6110 in the evolution of Mycobacterium tuberculosis. Tuberculosis (Edinburgh, Scotland) 87, 393–404.

    Article  PubMed  CAS  Google Scholar 

  6. Dale, J. W., Tang, T. H., Wall, S., Zainuddin, Z. F., and Plikaytis, B. (1997). Conservation of IS6110 sequence in strains of Mycobacterium tuberculosis with single and multiple copies. Tuber. Lung Dis. 78, 225–227.

    Article  CAS  Google Scholar 

  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.

    CAS  Google Scholar 

  8. Mathema, B., Kurepina, N. E., Bifani, P. J., and Kreiswirth, B. N. (2006). Molecular epidemiology of tuberculosis: current insights. Clin. Microbiol. Rev. 19, 658–685.

    Article  CAS  Google Scholar 

  9. Kurepina, N., Likhoshvay, E., Shashkina, E., Mathema, B., Kremer, K., van Soolingen, D., et al (2005). Targeted hybridization of IS6110 fingerprints identifies the W-Beijing Mycobacterium tuberculosis strains among clinical isolates. J. Clin. Microbiol. 43, 2148–2154.

    Article  CAS  Google Scholar 

  10. Bifani, P. J., Plikaytis, B. B., Kapur, V., Stockbauer, K., Pan, X., Lutfey, M. L., et al (1996). Origin and interstate spread of a New York City multidrug-resistant Mycobacterium tuberculosis clone family. JAMA 275, 452–457.

    Article  PubMed  CAS  Google Scholar 

  11. Bifani, P. J., Mathema, B., Liu, Z., Moghazeh, S.L., Shopsin, B., Tempalski, B., et al (1999). Identification of a W variant outbreak of Mycobacterium tuberculosis via population-based molecular epidemiology. JAMA 282, 2321–2327.

    Article  PubMed  CAS  Google Scholar 

  12. Bifani, P., Mathema, B., Campo, M., Moghazeh, S., Nivin, B., Shashkina, E., et al (2001). Molecular identification of streptomycin monoresistant Mycobacterium tuberculosis related to multidrug-resistant W strain. Emerg. Infect. Dis. 7, 842–848.

    CAS  Google Scholar 

  13. Mathema, B., Bifani, P. J., Driscoll, J., Steinlein, L., Kurepina, N., Moghazeh, S. L., et al (2002). Identification and evolution of an IS6110 low-copy-number Mycobacterium tuberculosis cluster. J. Infect. Dis. 185, 641–649.

    Article  CAS  Google Scholar 

  14. Heersma, H. F., Kramer, K., and van Embden, J. (1998). Computer analysis of IS6110 RFLP patterns of Mycobacterium tuberculosis, in Mycobacteria Protocols, Vol. 101, (Parish, T., and Stoker, N. G., eds.), Humana Press, London, pp. 395–422.

    Chapter  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Cite this protocol

Bifani, P., Kurepina, N., Mathema, B., Wang, XM., Kreiswirth, B. (2009). Genotyping of Mycobacterium tuberculosis Clinical Isolates Using IS6110-Based Restriction Fragment Length Polymorphism Analysis. In: Caugant, D. (eds) Molecular Epidemiology of Microorganisms. Methods in Molecular Biology™, vol 551. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-60327-999-4_14

Download citation

  • DOI: https://doi.org/10.1007/978-1-60327-999-4_14

  • Published:

  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-60327-998-7

  • Online ISBN: 978-1-60327-999-4

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics