| Summary: | Dental caries remains the major cause of loss of tooth function and associated morbidity. The development of new prevention and treatment measures requires a better understanding of the disease process, particularly the role of the associated microbiota. The aim of this study was to perform a comprehensive characterisation of the microbiota in dental caries using culture-independent and next generation sequencing methods, with a focus on improving the detection of bacteria with DNA of high G+C content. Oligonucleotide probes for taxa of interest were designed and evaluated using fluorescent in situ hybridisation (FISH). DNA extraction methods, PCR polymerases and primer pairs were compared for their ability to isolate and amplify 16S rRNA genes from species with low and high G+C content. DNA extraction method and polymerase were not found to significantly influence the detection of high G+C species but primer sequence was found to be of critical importance. Five “universal” PCR primer sets and culture were used to characterise the dentine caries microbiota of six subjects. From 3240 clones and isolates, 228 taxa representing eight phyla were identified by Sanger sequencing. Detection frequency of the high G+C Actinobacteria was 33% using culture, but 2.6-11.1% in the different clone libraries. The samples were analysed further by 454 pyrosequencing and 25758 sequences were identified to 264 taxa representing 11 phyla. Pyrosequencing allowed an analysis of greater depth, although the composition of the samples was comparable to those obtained by the Sanger-based method. Total and specific bacteria were detected successfully in excavated dentine by FISH with universal and specific probes, while in bisected teeth only total bacteria were seen with the universal probe. In conclusion, this study has revealed a highly diverse caries microbiota. Pyrosequencing increased detection of taxa and overall coverage, but all methods had associated biases which affected the results obtained.
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