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Journal of Clinical Microbiology, April 2003, p. 1637-1650, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1637-1650.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

PCR-Based Method To Differentiate the Subspecies of the Mycobacterium tuberculosis Complex on the Basis of Genomic Deletions

Richard C. Huard,1,2 Luiz Claudio de Oliveira Lazzarini,1 W. Ray Butler,3 Dick van Soolingen,4 and John L. Ho1*

Division of International Medicine and Infectious Diseases, Department of Medicine, Joan and Sanford I. Weill Medical College,1 Graduate School of Medical Sciences, Cornell University, New York, New York,2 Division of AIDS, STD and TB Laboratory Research, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia,3 National Institute of Public Health and the Environment, Bilthoven, The Netherlands4

Received 13 November 2002/ Returned for modification 8 January 2003/ Accepted 18 January 2003

The classical Mycobacterium tuberculosis complex (MtbC) subspecies include Mycobacterium tuberculosis, Mycobacterium africanum (subtypes I and II), Mycobacterium bovis (along with the attenuated M. bovis bacillus Calmette-Guérin [BCG]), and Mycobacterium microti; increasingly recognized MtbC groupings include Mycobacterium bovis subsp. caprae and "Mycobacterium tuberculosis subsp. canettii." Previous investigations have documented each MtbC subspecies as a source of animal and/or human tuberculosis. However, study of these organisms is hindered by the lack of a single protocol that quickly and easily differentiates all of the MtbC groupings. Towards this end we have developed a rapid, simple, and reliable PCR-based MtbC typing method that makes use of MtbC chromosomal region-of-difference deletion loci. Here, seven primer pairs (which amplify within the loci 16S rRNA, Rv0577, IS1561', Rv1510, Rv1970, Rv3877/8, and Rv3120) were run in separate but simultaneous reactions. Each primer pair either specifically amplified a DNA fragment of a unique size or failed, depending upon the source mycobacterial DNA. The pattern of amplification products from all of the reactions, visualized by agarose gel electrophoresis, allowed immediate identification either as MtbC composed of M. tuberculosis (or M. africanum subtype II), M. africanum subtype I, M. bovis, M. bovis BCG, M. caprae, M. microti, or "M. canettii" or as a Mycobacterium other than MtbC (MOTT). This MtbC PCR typing panel provides an advanced approach to determine the subspecies of MtbC isolates and to differentiate them from clinically important MOTT species. It has proven beneficial in the management of Mycobacterium collections and may be applied for practical clinical and epidemiological use.

* Corresponding author. Mailing address: Cornell University, Joan and Sanford I. Weill Medical College, Department of Medicine, Division of International Medicine and Infectious Diseases, Room A-421, 525 East 68th St., New York, NY 10021. Phone: (212) 746-6316. Fax: (212) 746-8675. E-mail: jlho{at}med.cornell.edu.

Journal of Clinical Microbiology, April 2003, p. 1637-1650, Vol. 41, No. 4
0095-1137/03/$08.00+0     DOI: 10.1128/JCM.41.4.1637-1650.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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