Analysis of microflora in dentinal tubule by denaturing gradient gel electrophoresis

Abstract

This study was performed to apply denaturing gradient gel electrophoresis to microbiological examination of endodontic infections. The method was used to assess the bacterial communities in dentinal tubules. Samples were collected using #15, #35, and #60 K-type files from five infected root canals during treatment. Deoxyribonucleic acid(DNA) was extracted from the samples, and 16S ribosomal DNA was amplified by PCR using universal primers. The polymerase chain reaction(PCR) products were separated in the denaturing gel and band patterns were compared between the deep (#60 file) and superficial layers (#15 file) of the dentin. The major bands were then excised and DNA fragments in the gel were cloned and sequenced. The sequence data were subjected to BLAST search in the GenBank database for determination of bacterial species or closest relatives. In three root canals, similar band patterns were observed in both superficial and deep layers of the dentin, while several specific bands remained in the deep layer in two roots. The bacteria isolated from the deep layers were Porphyromonas gingivalis, Olsenella profuse, Atopobium rimae, and Prevotella, Flexistipes, Firmicutes, Peptostreptococcus, Dialister, and Eubacterium spp. Unlike previous studies, gram-negative anaerobic rods were isolated from the deep layers. Clone library analysis was simultaneously performed and similar results were obtained. The method utilized here will be useful for microbiological examination of endodontic infections. In addition, although it is still unknown whether they were viable, this study demonstrated the presence of gram-negative rods in dentinal tubules.