Isothermal Amplification and Molecular Typing of the Obligate Intracellular Pathogen Mycobacterium leprae Isolated from Tissues of Unknown Origins

Abstract

Molecular diagnostic and epidemiology studies require appreciable amounts of high-quality DNA. Molecular epidemiologic methods have not been routinely applied to the obligate intracellular organism Mycobacterium leprae because of the difficulty of obtaining a genomic DNA template from clinical material. Accordingly, we have developed a method based on isothermic multiple-displacement amplification to allow access to a high-quality DNA template. In the study described in this report, we evaluated the usefulness of this method for error-sensitive, multiple-feature molecular analyses. Using test samples isolated from lepromatous tissue, we also evaluated amplification fidelity, genome coverage, and regional amplification bias. The fidelity of amplified genomic material was unaltered; and while regional differences in global amplification efficiency were seen by using comparative microarray analysis, a high degree of concordance of amplified genomic DNA was observed. This method was also applied directly to archived tissue specimens from leprosy patients for the purpose of molecular typing by using short tandem repeats; the success rate was increased from 25% to 92% without the introduction of errors. This is the first study to demonstrate that serial whole-genome amplification can be coupled with error-sensitive molecular typing methods with low-copy-number sequences from tissues containing an obligate intracellular pathogen.

Comparison of (GTG)5-oligonucleotide and ribosomal intergenic transcribed spacer (ITS)-PCR for molecular typing of Klebsiella isolates

“…Therefore, using MDA-amplified bacterial DNA derived from few bacterial cells may help to accomplish bacterial genotyping at reasonable costs and time. This is in accordance with similar studies were it was shown that MDA-amplified DNA derived from few bacterial cells and human biopsy specimens provided a reliable and representative source for multiple molecular typing analysis (Monstein et al, 2005;Groathouse et al, 2006;Ryberg et al, 2008;Tärnberg et al, 2009).…”

Molecular identification of blaSHV, blaLEN and blaOKP β-lactamase genes in Klebsiella pneumoniae by bi-directional sequencing of universal SP6- and T7-sequence-tagged blaSHV-PCR amplicons

“…By contrast, an automated nucleic extractor combined with multiple displacement amplification (MDA) of bacterial DNA yields DNA of high purity that can be used in multiple downstream applications. In recent years, MDA has been tried out for microbial and total DNA (bacterial and cellular) isolated from human biopsy specimens [20,21,23]. The present study shows the feasibility of using MDA-amplified bacterial DNA as a source in bla SHV, bla LEN and bla OKP genotyping analysis of K. pneumoniae clinical isolates having an ESBL-phenotype.…”

Whole‐community genome amplification (WCGA) leads to compositional bias in methane‐oxidizing communities as assessed by pmoA‐based microarray analyses and QPCR

“…caves (Gonzalez et al. , 2005), insects (Jeyaprakash and Hoy, 2004), human skin (Groathouse et al. , 2006), corals (Yokouchi et al.…”

“…Microbial communities have been analysed in, e.g. caves (Gonzalez et al, 2005), insects (Jeyaprakash and Hoy, 2004), human skin (Groathouse et al, 2006), corals (Yokouchi et al, 2006), subsurface and contaminated soils (Abulencia et al, 2006), marine viral assemblages (Angly et al, 2006) and contaminated groundwater (Wu et al, 2006). Communities that have been subjected to WCGA (Erwin et al, 2005;Kalyuzhnaya et al, 2006;Chen et al, 2008;Neufeld et al, 2008) include those involved in important biogeochemical functions, e.g.…”