DNA HYBRIDIZATION IN THE DIAGNOSIS OF INFECTIOUS DISEASE

DNA HYBRIDIZATION PROBES SPECIFIC FOR COMMON PATHOGENIC BACTERIA HAVE BEEN DEVELOPED. THE LONG TERM OBJECTIVE OF THIS RESEARCH IS THE PERFECTION OF SIMPLE PROTOCOLS AND A KIT USING DNA HYBRIDIZATION PROBES FOR THE DETECTION AND IDENTIFICATION OF PATHOGENS IN CLINICAL SPECIMENS. THE SPECIFIC AIM OF THIS PROPOSAL IS TO ASSESS THE FEASABILITY OF THIS APPROACH TO PATHOGEN IDENTIFICATION IN CLINICAL SPECIMENS BY: 1) DETERMINING NUMBERS AND METHODS OF RECOVERY OF PATHOGENS IN CLINICAL SPECIMENS; 2) ASSESSING THE FACILITY WITH WHICH THEY MAY BE LYSED AND THEIR DNA PREPARED FOR HYBRIDIZATION; 3) DETERMINING THE UTILITY OF PROCEDURES DEVELOPED FOR RAPID HYBRIDIZATION; AND 4) ASSESSING PROBE MODIFICATIONS TO ENHANCE DETECTABILITY. THE METHODS EMPLOYED INCLUDE CLONING AND IDENTIFICATION OF BACTERIAL DNA FRAGMENTS WITH UNIQUE BASE SEQUENCES, PROCEDURES FOR LYSIS OF BACTERIA AND RECOVERY OF DNA, PROTOCOLS FOR RAPID DNA-DNA HYBRIDIZATION, AND DETECTION OF HYBRID COMPLEXES. SUCCESSFUL COMPLETION OF PHASE I WILL DEMONSTRATE THAT DNA HYBRIDIZATIONS PROCEDURES CAN ACCURATELY AND QUICKLY DETECT AND IDENTIFY PATHOGENS IN CLINICAL MATERIAL. WITH FEASABILITY DEMONSTRATED, PHASE II DEVELOPMENT WILL REQUIRE THE CLONING AND TESTING OF ADDITIONAL PROBES SPECIFIC FOR PATHOGENS ENCOUNTERED IN INFECTIOUS DISEASE, THE MODIFICATION AND ADAPTATION OF PROCEDURES FOR DIFFERENT TYPES OF CLINICAL SPECIMENS AS WELL AS DEVELOPMENT OF ALTERNATIVE METHODS FOR DETECTION OF HYBRID COMPLEXES. THE APPLICATION OF RECOMBINANT DNA TECHNOLOGY TO DIAGNOSIS OF INFECTIOUS DISEASE REPRESENTS A FUNDAMENTAL DEPARTURE FROM CURRENT MICROBIOLOGICAL IDENTIFICATION PROCEDURES AND SHOULD PROVIDE UNSURPASSED ACCURACY IN ORGANISM IDENTIFICATION. DEVELOPMENT OF THIS PROCEDURE INTO A SIMPLE, RELIABLE KIT HAS MAJOR COMMERCIAL POTENTIAL.