title: 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.
authors.affiliation: Department of Microbiology, Immunology and Pathology, Colorado State University, B208 Microbiology Building, Fort Collins, CO 80523-1682, USA. richard.slayden@colostate.edu
authors.forename: Nathan A
authors.initials: NA
authors.lastname: Groathouse
authors.forename: Susan E
authors.initials: SE
authors.lastname: Brown
authors.forename: Dennis L
authors.initials: DL
authors.lastname: Knudson
authors.forename: Patrick J
authors.initials: PJ
authors.lastname: Brennan
authors.forename: Richard A
authors.initials: RA
authors.lastname: Slayden
mesh_terms: D015373:Bacterial Typing Techniques / Q000379:methods*; D004269:DNA, Bacterial / Q000032:analysis; D006801:Humans; D007918:Leprosy / Q000175:diagnosis / Q000382:microbiology; D009166:Mycobacterium leprae / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods
chemical_list: D004269:DNA, Bacterial