DNA PROBES FOR DIAGNOSIS OF GONOCOCCAL DISEASE

DEVELOPMENT OF AN EFFICACIOUS AND COST-EFFECTIVE DIAGNOSTIC TEST FOR NEISSERIA GONORRHOEAE INFECTION IS THE GOAL OF THIS PROJECT. ALTHOUGH CURRENT DIAGNOSTIC METHODS ARE SENSITIVE AND SPECIFIC, THEY ARE TIME CONSUMING AND COSTLY. BECAUSE OF THE ENORMOUS MORBIDITY OF GONOCOCCAL INFECTION IN THE U.S., THE POTENTIAL FOR COMMERCIAL APPLICATION OF SUCH A TEST IS EXCELLENT. THIS PHASE I PROPOSAL EXAMINES THE FEASIBILITY OF TWO DNA "PROBES" AS DIAGNOSTIC REAGENTS. ONE WILL BE BASED ON THE DNA SEQUENCE OF A 26 BASEPAIR REPETITIVE ELEMENT FOUND IN THE GENOMES OF NEISSERIA SPP. A 21 BASE OLIGONUCLEOTIDE WILL BE COMMERCIALLY SYNTHESIZED AND ENZYMATICALLY BIOTINYLATED. THE SECOND PROBE WILL BE A SINGLE STRANDED DNA FRAGMENT OF THE GONOCOCCAL OUTER MEMBRANE PROTEIN III GENE. THIS PROBE WILL BE BIOTINYLATED CHEMICALLY. A RESTRICTION FRAGMENT OF THIS GENE DEMONSTRATING THE BEST SPECIES SPECIFICITY WILL BEDETERMINED BY HYBRIDIZATION ANALYSIS WITH GENOMIC DNA FROM SEVERAL NEISSERIA SPP. BOTH PROBES WILL BE EXAMINED FOR SENSITIVITY USING PURIFIED GONOCOCCAL DNA AND HYBRIDIZATION ANALYSIS ON NITROCELLULOSE. BIOTINYLATED DNA PROBES WILL BE DETECTED WITH COMPLEXES OF STREPTAVIDIN AND BIOTINYLATED ALKALINE PHOSPHATASE OR UREASE. PROBES WILL BE CONSIDERED FOR FURTHER DEVELOPMENT (PHASE II), IF THEIR LEVEL OF SENSITIVITY ALLOWS DETECTION OF LESS THAN TEN THOUSAND GONOCOCCI WITH INSIGNIFICANT BACKGROUND "NOISE".