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Rapid variable-number tandem-repeat genotyping for Mycobacterium leprae clinical specimens.

Abstract

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.

More information

Type
Journal Article
Author
Kimura M
Sakamuri RM
Groathouse NA
Rivoire BL
Gingrich D
Krueger-Koplin S
Cho S
Brennan PJ
Vissa V
Year of Publication
2009
Journal
Journal of clinical microbiology
Volume
47
Issue
6
Number of Pages
1757-66
Date Published
2009 Jun
Language
eng
ISSN Number
1098-660X
DOI
10.1128/JCM.02019-08
Alternate Journal
J. Clin. Microbiol.
Publication Language
eng