DNA Fingerprinting of <em>Mycobacterium leprae</em> Strains Using Variable Number Tandem Repeat (VNTR) - Fragment Length Analysis (FLA)

Abstract

The study of the transmission of leprosy is particularly difficult since the causative agent, Mycobacterium leprae, cannot be cultured in the laboratory. The only sources of the bacteria are leprosy patients, and experimentally infected armadillos and nude mice. Thus, many of the methods used in modern epidemiology are not available for the study of leprosy. Despite an extensive global drug treatment program for leprosy implemented by the WHO 1 , leprosy remains endemic in many countries with approximately 250,000 new cases each year.2 The entire M.

Mycobacterium leprae in Colombia described by SNP7614 in gyrA, two minisatellites and geography

“…The identification of multi-case family and community clusters indicates delay in diagnosis and continued transmission; these situations can be addressed locally and nationally. Secondary and higher level genotyping should then be possible due to the growing number of VNTR and SNP markers (Kimura et al, 2009, Jensen et al, 2011). There are other alternatives for genotyping which are not dependent on DNA sequencing methods such as the high resolution melt-PCR (HRM-PCR); as applied here for the SNP typing of a subset of samples (Li et al, 2012) and SNP typing by PCR-RFLP (Sakamuri et al, 2009).…”

Genotyping comparison ofMycobacterium lepraeisolates by VNTR analysis from nasal samples in a Brazilian endemic region

“…The NHDP63 VNTR and amplicon size were verified through gene sequencing. The amplicon sizes at each locus for the positive control were 384 bp to AC8b, 207 bp to AC9, 125 bp to AC8a and 307 bp to GTA9 [4,9]. PCR amplifications were performed using using the HotStartTaq Master Mix (Qiagen, Hilden, Germany), 0.2 μM of each primer pair and 2 μl of template DNA.…”

“…PCR amplifications were performed using using the HotStartTaq Master Mix (Qiagen, Hilden, Germany), 0.2 μM of each primer pair and 2 μl of template DNA. Thermocycling conditions used were: initial denaturation at 95 °C for 15 min followed by 40 cycles of 94 °C for 30s, 60 °C for 90s, 72 °C for 90s followed by a final elongation step f 72 °C for 10 m [4,9].…”

“…Allele copy number was determined by denaturation of amplicons and capillary gel electrophoresis on the sequencer ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA). Capillary electrophoresis (length 50 cm, polymer POP-7) was conducted at 15 kV over 60 °C with a running time of 45 min [4,9]. Copy number definition of each of the four allele were defined using the Peak Scanner software (Applied Biosystems, Foster City, CA).…”

“…Variable number tandem repeat (VNTR) provides data about the pattern of variation in the Mycobacterium leprae genome [3][4][5]. The VNTR typing tool is based on the number of repetitive sequences in polymorphic microand mini-satellite regions of the bacteria [6].…”