In Phase I, a new method of directly conjugating peroxidase to nucleic acids was investigated in detail. The feasibility of using probes labeled in this manner to detect pathogen sequences was demonstrated, using both filter and in situ hybridizations. In Phase II we propose to refine these methods and to apply them to the diagnosis of pathogens of clinical importance. Our goal is either to reduce drastically, the time required for definitive tissue culture diagnosis, or to eliminate the requirement for culture completely. Our Phase I study has generated significant interest in the research community. Collaborations have been established with expert consultants who will supply DIGENE with molecular cloned DNA probes, advice, and clinical samples of HSV, EBV, CMV, Influenza and Rhinovirus to facilitate our Phase II work. Probes against two pathogens--one DNA virus and one RNA virus--will be chosen from this list and prototype clinical tests to diagnose these infections will be developed. The pathogens chosen will be determined by: (a) the experimental results in the first half of Phase II and; (b) the extent to which the use of the DIGENE technique will result in an improved clinical outcome and therapy.
