Enumeration of prominent oral bacteria to aid in assessing caries and periodontal diseases requires a reliable, quantitative, sensitive and specific diagnostic test. Nucleic acid probes provide a reliable and specific diagnostic tool but may require signal amplification for necessary sensitivity. OmniGene has developed a unique amplification system called Ligase Chain Reaction (LCR) whose performance compares favorably with other available amplification methods, such as Polymerase Chain Reaction (PCR). The feasibility of using LCR amplification protocols to detect oral bacteria has been demonstrated using a cloned DNA probe which was previously identified at OmniGene, and shown to be specific for Actinobacillus actinomycetemcomitans. In this Phase I proposal species- specific DNA probes will be identified for six supraginigival organisms. The use of LCR amplification requires well-characterized and specific oligonucleotide probes. In Phase I, whole genomic DNA will be isolated from the six target strains and DNA libraries of each constructed. The cross-reactivity between the target species and other oral bacteria will be determined using chromosomal DNA as probes. The identification of the most cross-reactive species will enable an efficient screening protocol to be designed for screening the libraries in order to isolate cloned species- specific probes. In a Phase II proposal, the cloned probes isolated in Phase I will be sequenced, appropriate diagnostic target regions identified, and LCR probe sets synthesized. The probe sets will be tested in the LCR format and reaction conditions defined in order to maximize the specificity and sensitivity of the system. This will include designing a sample preparation protocol which provides DNA with sufficient purity to permit LCR amplification and implementing a non-isotopic read-out format for handling multiple samples in a short time. Finally, field testing required for regulatory review would be initiated.
