Análise microbiológica de infecções endodônticas utilizando a associação das técnicas do Multiple Displacement Amplification (MDA) e da Hibridização DNA-DNA (Checkerboard)

Abstract

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. 66 samples were collected from teeth with endodontic infections. Non-amplified and MDA 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 determined using the Wilcoxon signed ranks test and adjusted for multiple comparisons. The average amount of DNA present in clinical samples ranged from 6.80 (± 5.2) ng before to 6.26 (± 1.73) ìg after MDA. 70 of the 77 DNA probes hybridized with one or more of the non-amplified samples, while 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 ranging from 89-100% of samples. The mean number (± SEM) of species at counts >104 in amplified samples was 51.2 ± 2.2 and in non-amplified samples was 14.5 ± 1.7. The combination of MDA and checkerboard DNA-DNA hybridization demonstrated the presence of wide range of bacterial species in endodontic samples and could facilitate studies evaluating the microbiota of endodontic infections.