SEARCHING FOR NEW PERIODONTAL PATHOGENS: INSIGHTS FROM AN ENHANCED RNA-OLIGONUCLEOTIDE QUANTIFICATION TECHNIQUE (ROQT)

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  • March 22, 2019
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  • Herrera, Bruno
    • Affiliation: School of Dentistry, Department of Periodontology
Abstract
  • Aims: Fifty percent of the oral microbiome remains unrecognized or uncultured. In order to study this segment of the microbiota, we previously developed the RNA-Oligonucleotide Quantification Technique (ROQT). The aim of this study was to optimize the ROQT technique regarding its sensitivity, specificity and cost-effectiveness. Material and Methods: Total nucleic acids (TNA) were extracted from bacterial suspensions and subgingival biofilm samples using manual and an automated protocol. RNA, DNA and Locked Nucleic Acid (LNA) digoxigenin-labeled oligonucleotide probes targeting 21 cultured/uncultured taxa were synthesized. Tests were performed using 10ng TNA, 106 bacterial cells, and RNA and DNA standards for quantification. Probe specificity was determined by targeting 96 oral bacterial species; sensitivity was assessed using serial dilutions of reference bacterial strains. The tested conditions were assessed in a small pilot study with subgingival biofilm samples. Significance of differences between test conditions and subject groups was determined using the Mann–Whitney U-test. Results: LNA-oligunucleotides probes yielded stronger signals without cross-reactions, when compared with DNA and RNA oligonucleotides probes. The automated method at 63C consistently yields stronger signals in comparison to the manual protocol. Samples from patients with periodontitis showed higher levels of subgingival bacteria based on the universal probe signals than samples from periodontally healthy subjects. Overall, the most commonly detected uncultivated/unrecognized species in the samples from severe sites were probe Treponema sp ot254, TM7 ot356, Fretibacterium sp ot360, Y73 Selenomonas sp ot134, 54, and Desulfobulbus sp. ot041. In the cultivated segment of the microbiota, the most abundant taxa were Tannerella forsythia ot613, L66 Oribacterium sp ot78, K76 Bacterioidetes sp ot274, Fretibacterium fastidiosum ot363 and Porphyromonas gingivalis ot619. Conclusion: The use of automated TNA extraction, LNA probes and RNA standards enhances the sensitivity, specificity, throughput, and cost-effectiveness of ROQT.
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Advisor
  • Offenbacher, Steven
  • Teles, Flavia
  • Marchesan, Julie
Degree
  • Master of Science
Degree granting institution
  • University of North Carolina at Chapel Hill
Graduation year
  • 2018
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