FLUORESCENCE IN-SITU HYBRIDIZATSON WITH COSMID CLONES FOR THE DETECTION OF HUMAN CYTOMEGALOVIRUS DNA IN PERIPHERAL-BLOOD LEUKOCYTES

Radioactive in situ hybridization techniques or enzymatic detection pr ocedures of hapten-modified human cytomegalovirus (HCMV) probes have b een widely used for studying the infection of peripheral blood leukocy tes with HCMV. This report describes significant improvements in terms of signal resolution which can be obtained by applying a highly sensi tive fluorescence in situ hybridization (FISH) technique in conjunctio n with a large subgenomic HCMV DNA probe. Three cosmid clones spanning 119.1 kb of the HCMV genome (230 kb) were used to construct the digox igenin-11-dUTP-labeled probe which was found to be superior to a total HCMV probe representing the entire genome. Crucial hybridization para meters were analyzed systematically in order to ensure optimal resolut ion power and sensitivity. The protocol was successfully applied to HC MV-infected fibroblasts and peripheral blood leukocytes of 12 transpla nt patients and unambiguously facilitated the precise intracellular lo calization of HCMV genomes in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any back ground staining, we recommend the use of this protocol as a sensitive approach for further virological analyses of the interactions between HCMV and peripheral blood leukocytes at the single-cell level.