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Journal of Clinical Microbiology, June 2009, p. 1757-1766, Vol. 47, No. 6
0095-1137/09/$08.00+0     doi:10.1128/JCM.02019-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Rapid Variable-Number Tandem-Repeat Genotyping for Mycobacterium leprae Clinical Specimens{triangledown}

Miyako Kimura,1 Rama Murthy Sakamuri,1 Nathan A. Groathouse,1 Becky L. Rivoire,1 David Gingrich,2 Susan Krueger-Koplin,2 Sang-Nae Cho,3 Patrick J. Brennan,1 and Varalakshmi Vissa1*

Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, Colorado,1 Macromolecular Resources, Colorado State University, Fort Collins, Colorado,2 Department of Microbiology, College of Medicine, Yonsei University, Seoul, Republic of Korea3

Received 18 October 2008/ Returned for modification 6 March 2009/ Accepted 13 April 2009

Mycobacterium leprae is the noncultivable pathogen of leprosy. Since the genome sequence of an isolate of M. leprae has become available, multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been explored as a tool for strain typing and identification of chains of transmission of leprosy. In order to discover VNTRs and develop methods transferable to clinical samples, MLVA was applied to a global collection of M. leprae isolates derived from leprosy patients and propagated in armadillo hosts. PCR amplification, agarose gel electrophoresis, and sequencing methods were applied to DNA extracts from these infected armadillo tissues (n = 21). We identified polymorphisms in 15 out of 25 short-tandem-repeat (STR) loci previously selected by in silico analyses of the M. leprae genome. We then developed multiplex PCR for amplification of these 15 loci in four separate PCRs suitable for fluorescent fragment length analysis and demonstrated STR profiles highly concordant with those from the sequencing methods. Subsequently, we extended this method to DNA extracts from human clinical specimens, such as skin biopsy specimens (n = 30). With these techniques, mapping of multiple loci and differentiation of genotypes have been possible using total DNA extracts from limited amounts of clinical samples at a reduced cost and with less time. These practical methods are therefore available and applicable to answer focused epidemiological questions and to allow monitoring of the transmission of M. leprae in different countries where leprosy is endemic.

* Corresponding author. Mailing address: Department of Microbiology, Immunology, and Pathology, Colorado State University, Fort Collins, CO 80523. Phone: (970) 491-0752. Fax: (970) 491-1815. E-mail:vvissa{at}colostate.edu

{triangledown} Published ahead of print on 22 April 2009.

Journal of Clinical Microbiology, June 2009, p. 1757-1766, Vol. 47, No. 6
0095-1137/09/$08.00+0     doi:10.1128/JCM.02019-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Sakamuri, R. M., Kimura, M., Li, W., Kim, H.-C., Lee, H., Kiran, M. D., Black, W. C. IV, Balagon, M., Gelber, R., Cho, S.-N., Brennan, P. J., Vissa, V. (2009). Population-Based Molecular Epidemiology of Leprosy in Cebu, Philippines. J. Clin. Microbiol. 47: 2844-2854 [Abstract] [Full Text]  


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