DETECT. OF INFECT. DIS. WITH ENZYME-LABELED DNA PROBES

THIS PROJECT WILL CREATE FASTER AND MORE SENSITIVE DNA PROBEASSAYS UTILIZING THE DRAMATICALLY INCREASED SENSITIVITY THATCAN BE OBTAINED WITH CHEMILUMINESCENT SUBSTRATES FOR ALKALINE PHOSPHATASE USED IN PLACE OF CONVENTIONAL COLORIMETRIC SUBSTRATES IN ENZYME-BASED DNA PROBE ASSAYS. IN PHASE I, AN EVALUATION OF THE PERFORMANCE OF A MOLECULAR BIOSYSTEMS SNAP DNA TEST KIT SPECIFIC FOR HERPES SIMPLEX 1 VIRUS WITH THE CHEMILUMINESCENT SUBSTRATES 3-(2'-SPIRO-ADAMANTANE)-4-(3'-PHOSPHORYLOXY) PHENYL-1,2-DIOXETANE (AMPPD) IS PLANNED. RAPID DETECTION OF LESS THAN 10(5) COPIES OF TARGET DNA IS ANTICIPATED BY IMAGING THE CHEMILUMINESCENT SIGNAL ON INSTANT PHOTOGRAPHIC FILM. ALSO INCORPORATED INTO THIS RESEARCH IS A PROGRAM TO DECREASE THE NONSPECIFIC BINDING LEVELS THAT LEAD TO SENSITIVITY LIMITING BACKGROUND SIGNAL. THIS BACKGROUND, MOST LIKELY, IS DUE TO ENDOGENOUS ALKALINE PHOSPHATASE IN ALBUMIN PREPARATIONS AND BUFFER IMPURITIES. PHASE II OF THIS PROJECT WILL INVOLVE THE APPLICATION OF THERESULTS OF PHASE I TO IN SITU DETECTIONS OF VIRAL INFECTIONSIN CELLS AND TISSUES. CHEMILUMINESCENCE SHOULD ALLOW THE DETECTION OF INDIVIDUAL INFECTED CELLS WITHOUT THE USE OF A MICROSCOPE. IN ADDITION, THE DEVELOPMENT OF A DISPOSABLE IMAGING DEVICE THAT CAN BE USED TO IMAGE RESULTS FROM CHEMILUMINESCENT DNA PROBE ASSAYS FOR INFECTIOUS DISEASES ISPLANNED.