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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
World Health Organization (2007). Tuberculosis Facts. Available at: http://www.who.int/tb/publications/2007/factsheet_2007.pdf.
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.
Mahillon, J., and Chandler, M. (1998). Insertion sequences. Microbiol. Mol. Biol. Rev. 62, 725–774.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights 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