CYTOMEGALOVIRUS DETECTION USING NONRADIOACTIVE DNA PROBES IN SITU

CYTOMEGALOVIRUS (CMV), A MEMBER OF THE HERPES VIRUS GROUP, IS PRESENT IN THE BLOOD OF A LARGE PORTION OF THE HUMAN POPULATION. ADMINISTRATION OF THIS BLOOD TO HIGH-RISK PATIENTS MAY RESULT IN DEATH. ENZO PROPOSES THE DEVELOPMENTOF A QUICK, SPECIFIC, EASY AND ECONOMICAL DIAGNOSTIC TEST, BASED ON NONRADIOACTIVE DNA-DNA HYBRIDIZATION, TO DIRECTLY SCREEN HUMAN BLOOD FOR CMV. PHASE I DEVELOPMENT WILL BE PERFORMED USING CMV-INFECTED HUMAN FIBROBLAST MONOLAYERS MIXED WITH HUMAN BLOOD OR BUFFY COAT LEUKOCYTES AS A MODEL SYSTEM. DETECTION OF VIRAL DNA WILL BE BASED ON IN SITU HYBRIDIZATION OF NONRADIOACTIVE BIOTINYLATED CMV-SPECIFIC DNA PROBES. VISUALIZATION WILL INVOLVE DETECTION OF BIOTIN BASED ON ENZYMATIC OR FLUORESCENT METHODS. THIS MODEL WILL ALLOW FOR OPTIMIZATION OF SAMPLE TREATMENT AND HYBRIDIZATION-DETECTION CONDITIONS IN ORDER TO ATTAIN A RELIABLE AND STRAIGHTFORWARD DIAGNOSTIC ASSAY THAT CAN BE PERFORMED IN THE TYPICAL CLINICAL OR BLOOD LABORATORY. PHASE II WILL INVOLVE TESTING THIS DIRECT ASSAY ON HUMAN BLOOD SAMPLES. THE RESULTS WILL BE COMPARED WITH THOSE OBTAINED FROM STANDARD CELL CULTURE ASSAYS.