DNA PROBE ASSAY FOR B. BURGDORFERI--LYME DISEASE

LYME DISEASE, A MULTISYSTEMIC DISORDER, BEGINS WITH THE TRANSMISSION OF THE BACTERIUM, BORRELIA BURGDORFERI, AT THE SITE OF A TICK BITE AND PROGRESSES IN STAGES TO PRESENT DERMATOLOGICAL, NEUROLOGICAL, AND ARTHRITIC SYNDROMES. THE OBJECTIVES OF THIS PROJECT ARE TO: (1) DEVELOP A COMMERCIALLY VIABLE DIAGNOSTIC DNA PROBE ASSAY IN KIT FORM FOR THE DETECTION OF B. BURGDORFERI IN BLOOD, AND (2) EVALUATE THE ASSAY BY USING CLINICAL SAMPLES FROM PATIENTS WITH LYME DISEASE. SPECIES-SPECIFIC DNA PROBES COMPLEMENTARY TO VARIABLE DOMAINS OF THE B. BURGDORFERI RIBOSOMAL RNA HAVE ALREADY BEEN DEVELOPED. TO INCORPORATE THESE PROBES INTO A WORKABLEAND REPRODUCIBLE ASSAY IN KIT FORM, A SOLUTION HYBRIDIZATIONFORMAT USING TWO PROBES, A RADIOLABELED REPORTER PROBE AND ABIOTINYLATED CAPTURE PROBE, HAS BEEN DEVISED. CLINICAL SAMPLES ARE TREATED WITH GUANIDINE ISOTHIOCYANATE, WHICH SOLUBILIZES BACTERIA AND CELLS, PROTECTS THE RNA FROM DEGRADATION, AND IMPROVES HYBRIDIZATION KINETICS. STREPTAVIDIN-COATED MAGNETIC BEADS ARE USED AS A SOLID PHASETO IMMOBILIZE THE COMPLEXES. A DNA PROBE TEST FOR B. BURGDORFERI WILL ALLOW FOR THE EARLYDIAGNOSIS OF LYME DISEASE AND HENCE THE INITIATION OF ANTIBIOTIC TREATMENT BEFORE CHRONIC SEQUELAE DEVELOP. THE TEST WILL ALSO IMPROVE IDENTIFICATION OF ENDEMIC FOCI BY PUBLIC HEALTH PROFESSIONALS.