The broad objective of the proposed research is to produce specific and sensitive DNA probes for the rapid identification and enumeration of microorganisms involved in the pathogenesis of periodontal diseases. This proposal will focus on developing DNA probes to bacteria in four groups of gram negative rods that have been implicated in periodontal diseases. These groups contain the following genera: 1) Campylobacter and Wolinella, 2) Actinobacillus, Haemophilus, and Pasteurella, 3) Eikenella, and 4) Bacteroides.
The Specific Aims consist of the four logical steps required to develop DNA probes to bacterial 16S ribosomal RNA.
The first Aim i s to determine the sequence of the 16S rRNA for species in the selected groups. Sequences will be determined using a modification of the Sanger dideoxy chain termination sequencing method in which reverse transcriptase is used to elongate primers complementary to conserved regions of 16S rRNA.
The second Aim i s to design and construct complementary DNA probes to 16S rRNA by identifying unique 24-30 base regions of aligned sequences. Radiolabeled and enzyme-linked probes will be synthesized for the oral organisms in each of the four selected bacterial groups.
The third Aim i s to validate the specificity and sensitivity of these probes in hybridization studies using purified RNA, reference pure cultures and cultured clinical isolates. Probe identification of bacteria will be compared to conventional biochemical characterization. The last Aim is to validate the probes for identification and enumeration of target organisms directly in clinical samples without in vitro cultivation. These results will be compared to conventional identification and enumeration methods. The development of DNA probes for the rapid identification and enumeration of periodontal pathogens will aid in the study, diagnosis and treatment of periodontal diseases.
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