The broad objectives of the proposed research are to develop methods for 1) the rapid detection and enumeration of specific oral pathogens and 2) the hierarchal identification of any oral isolate. To achieve these objectives, 16s rRNA based DNA probes will be developed. This proposal will focus on developing DNA- probes to oral bacteria within four target groups: Campylobacter/Wolinella; Fusobacterium; Bacteroides/Prevotella/Porphyromonas; and Selenomonas/Veillonella. A fifth set of probes will be developed to higher taxa for the hierarchal identification of isolates.
The first Aim i s to determine 16S rRNA sequences for strains relevant to probe design. Proper validation of probes requires determination of intraspecies variation.
The second Aim i s to design and fully validate DNA probes for identification of phylum, family, genus, species and subspecies. These probes will be used to detect specific periodontal pathogens, and will be used hierarchically to identify and classify unknown oral isolates. Probe specificity will be validated using a panel of 252 oral strains. Probe sensitivity will be validated by testing 100 strains of the target species or taxa.
The third Aim i s to develop methods for use of nonradioactively labeled 16S rRNA based DNA probes. Use of nonradioactive probes provides for a safer workplace and will foster the transfer of these probes to other research laboratories.
The fourth Aim i s to develop methods for direct bacterial enumeration from clinical samples without In vitro cultivation. Fluorescently labeled probes will be used for epifluorescence microscopy, and PCR technology will be used to amplify target DNA of bacteria present in concentrations below the current limit of detection. This research will enhance our ability to detect, identify and enumerate oral pathogens. Hierarchal use of taxa-specific probes represents a novel approach for the identification of oral species. Validated probes from the proposed research will be valuable in studies of oral bacterial ecology, the effect of periodontal therapies on persistence and recolonization, and the role of periodontal pathogens in infections elsewhere in the body.
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