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

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TitleRapid variable-number tandem-repeat genotyping for Mycobacterium leprae clinical specimens.
Publication TypeJournal Article
AuthorsKimura M, Sakamuri RM, Groathouse NA, Rivoire BL, Gingrich D, Krueger-Koplin S, Cho S-N, Brennan PJ, Vissa V
Abbrev. JournalJ. Clin. Microbiol.
JournalJournal of clinical microbiology
Year of Publication2009
Volume47
Issue6
Pagination1757-66
Publication Languageeng
KeywordsAnimals, Armadillos, Bacterial Typing Techniques, DNA Fingerprinting, DNA, Bacterial, Genotype, Humans, Leprosy, Minisatellite Repeats, Molecular Epidemiology, Mycobacterium leprae, Polymorphism, Genetic, Time Factors
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.

PubMed URL

http://www.ncbi.nlm.nih.gov/pubmed/19386839?dopt=Abstract

DOI10.1128/JCM.02019-08
Link to full texthttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2691099/pdf/2019-08.pdf
PubMed Central IDPMC2691099
Grant ListR01 AI063457 / AI / NIAID NIH HHS / United States
R03 AI059644 / AI / NIAID NIH HHS / United States
AI-063457 / AI / NIAID NIH HHS / United States
AI-47197 / AI / NIAID NIH HHS / United States
N01AI25469 / AI / NIAID NIH HHS / United States
N0I AI-25469 / AI / NIAID NIH HHS / United States
AI-59644 / AI / NIAID NIH HHS / United States
R01 AI047197 / AI / NIAID NIH HHS / United States

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