02633nas a2200397 4500000000100000008004100001260001300042653001200055653001500067653003200082653002300114653001900137653001300156653001100169653001200180653002600192653002700218653002500245653002600270653001700296100001300313700001600326700001700342700001400359700001500373700002100388700001000409700001500419700001200434245009600446856007300542300001200615490000700627520158700634022001402221 2009 d c2009 Jun10aAnimals10aArmadillos10aBacterial Typing Techniques10aDNA Fingerprinting10aDNA, Bacterial10agenotype10aHumans10aleprosy10aMinisatellite Repeats10aMolecular Epidemiology10aMycobacterium leprae10aPolymorphism, Genetic10aTime Factors1 aKimura M1 aSakamuri RM1 aGroathouse N1 aRivoire B1 aGingrich D1 aKrueger-Koplin S1 aCho S1 aBrennan PJ1 aVissa V00aRapid variable-number tandem-repeat genotyping for Mycobacterium leprae clinical specimens. uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC2691099/pdf/2019-08.pdf a1757-660 v473 a
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.
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