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J. Clin. Microbiol. doi:10.1128/JCM.02618-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The use of multiple displacement amplification and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections

Department of Periodontology, The Forsyth Institute, Boston MA; Federal University of Minas Gerais - School of Dentistry, Belo Horizonte, Minas Gerais, Brazil; Department of Oral Medicine, Infection and Immunity, Harvard School of Dental Medicine, Boston, MA, USA

^* To whom correspondence should be addressed. Email: luitauna{at}yahoo.com.br.

	Abstract

Introduction: Multiple Displacement Amplification (MDA) has been used to uniformly amplify bacterial genomes present in small samples, providing abundant targets for molecular analysis. The purpose of this investigation was to combine MDA and checkerboard DNA-DNA hybridization to examine the microbiota of endodontic infections.

Methods: 66 samples were collected from teeth with endodontic infections. Non-amplified and amplified samples were analyzed by checkerboard DNA-DNA hybridization for levels and proportions of 77 bacterial taxa. Counts, % DNA probe counts and % of teeth colonized for each species in amplified and non-amplified samples were computed. Significance of differences for each species between amplified and non- amplified samples was sought with Wilcoxon signed ranks test and adjusted for multiple comparisons.

Results: The amount of DNA in the samples ranged from 6.80 (± 5.2) ng before to 6.26 (± 1.73) µg after MDA. 70 of the 77 DNA probes hybridized with one or more of the non-amplified samples. All probes hybridized with at least one sample after amplification. Most commonly detected species at levels > 10⁴ in both amplified and non-amplified samples were Prevotella tannerae and Acinetobacter baumannii at frequencies between 89-100% of samples. The mean number of species at counts >10⁴ in amplified samples was 51.2 ± 2.2 and in non-amplified samples was 14.5 ± 1.7.

Conclusions: The endodontic microbiota was far more complex than previously shown, although microbial profiles at teeth with or without periradicular lesions did not differ significantly. Species commonly detected in endodontic samples included P. tannerae, Prevotella oris and A. baumannii.