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.

Figures

FIG. 1.

Gel electrophoresis and FLA of 15 VNTR loci from five Cebu, the Philippines, clinical isolates amplified using the combination 1, 2, 3, and 4 primer sets. (Aa, Ba, Ca, and Da) Gel electrophoresis of multiplex-PCR products (combinations 1 to 4). Lanes: M, 20-bp DNA marker (Bio-Rad); P, multiplex-PCR products from NHDP63 (positive control); 28, 32, 35, 39, and 42, multiplex-PCR products from clinical isolates; N, negative control. (Ab, Bb, Cb, and Db) Electropherograms obtained from FLA, where the orange peaks represent the LIZ-500 size standards and red, blue, black, and green peaks represent the VNTR products in each of the combinations. Arrows indicate the peaks selected as the allele for each locus.

FIG. 1.

Gel electrophoresis and FLA of 15 VNTR loci from five Cebu, the Philippines, clinical isolates amplified using the combination 1, 2, 3, and 4 primer sets. (Aa, Ba, Ca, and Da) Gel electrophoresis of multiplex-PCR products (combinations 1 to 4). Lanes: M, 20-bp DNA marker (Bio-Rad); P, multiplex-PCR products from NHDP63 (positive control); 28, 32, 35, 39, and 42, multiplex-PCR products from clinical isolates; N, negative control. (Ab, Bb, Cb, and Db) Electropherograms obtained from FLA, where the orange peaks represent the LIZ-500 size standards and red, blue, black, and green peaks represent the VNTR products in each of the combinations. Arrows indicate the peaks selected as the allele for each locus.