DNA PROBES FOR THE DIAGNOSIS OF PERIODONTAL PATHOGENS

THE ROLE OF SPECIFIC BACTERIA AS THE ETIOLOGIC AGENTS OF GINGIVITIS AND PERIODONTITIS IS SUPPORTED BY A GROWING SCIENTIFIC LITERATURE. HOWEVER, CONVENTIONAL MICRO- BIOLOGICAL TECHNIQUES HAVE RESTRICTED ADVANCES IN DISEASE DIAGNOSIS AND MONITORING. THE PURPOSE OF THIS PHASE I PROJECT IS TO DEVELOP DNA PROBES FROM THE CHROMOSOMAL DNA OFBACTERIA ASSOCIATED WITH GINGIVITIS AND PERIODONTITIS. THE PROBES WILL BE LABELED WITH 32P AND HYBRIDIZED AGAINST BACTERIAL DNA IMMOBILIZED ON NITROCELLULOSE. THE HYBRID- IZATION SPECIFICITY OF EACH PROBE WILL BE EVALUATED IN THE STANDARD NACL SOLUTION, AND IN A NEW SALT SOLUTION, TETRAETHYLAMMONIUM CHLORIDE (TEACL). THIS NEW SALT SYSTEM ABOLISHES THE PREFERENTIAL MELTING OF A-T VERSUS G-C BASE PAIRS IN DNA HYBRIDS, THUS PRODUCING A SIGNIFICANT SHARPENING OF THE MELTING PROFILE (<1C IN TEACL VS. 5-10C INNACL) AND ENHANCING THE SPECIFICITY OF THE PROBES. DNA PROBES IN A TEACL SALT SYSTEM WILL ALLOW THE DEVELOPMENT OF HIGHLY SENSITIVE AND SPECIFIC DNA PROBE ASSAYS FOR THE DETECTION, ENUMERATION, AND MONITORING OF PATHOGENIC BACTERIA. THE LONG-TERM OBJECTIVE OF THIS PROJECT IS TO PROVIDE A SIMPLE, RAPID, ACCURATE, AND LOW-COST METHODOLOGY FOR THE DETECTION AND IDENTIFICATION OF BACTERIA PRESENT IN CLINICAL ISOLATES FROM PATIENTS WITH GINGIVITIS OR PERIO- DONTITIS. DIAGNOSTIC TESTS FOR SPECIFIC PATHOGENS WILL BE DEVELOPED AND PRODUCED COMMERCIALLY FOR USE BY CLINICAL DIAGNOSTIC LABORATORIES AND IN THE DENTAL OFFICE.