Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalovirus DNA in peripheral blood leukocytes.
Radioactive in situ hybridization techniques or enzymatic detection procedures of hapten-modified human cytomegalovirus (HCMV) probes have been widely used for studying the infection of peripheral blood leukocytes with HCMV. This report describes significant improvements in terms of signal resolution which can be obtained by applying a highly sensitive fluorescence in situ hybridization (FISH) technique in conjunction 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 digoxigenin-11-dUTP-labeled probe which was found to be superior to a total HCMV probe representing the entire genome. Crucial hybridization parameters were analyzed systematically in order to ensure optimal resolution power and sensitivity. The protocol was successfully applied to HCMV-infected fibroblasts and peripheral blood leukocytes of 12 transplant patients and unambiguously facilitated the precise intracellular localization of HCMV genomes in infected cells. Because of its excellent resolution properties, accompanied by the virtual absence by any background 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.
Hackstein
H
H
Institute of Immunology and Transfusion Medicine, University of Lübeck School of Medicine, Germany.
Jahn
G
G
Kirchner
H
H
Bein
G
G
D003001:Cloning, Molecular; D003360:Cosmids; D003587:Cytomegalovirus / Q000302:isolation & purification*; D003586:Cytomegalovirus Infections / Q000821:virology; D015342:DNA Probes; D004276:DNA, Satellite; D004279:DNA, Viral / Q000032:analysis*; D005347:Fibroblasts / Q000821:virology; D005821:Genetic Techniques; D006801:Humans; D007124:Immunoenzyme Techniques; D017404:In Situ Hybridization, Fluorescence; D007962:Leukocytes / Q000821:virology*; D016377:Organ Transplantation; D012680:Sensitivity and Specificity
D015342:DNA Probes; D004276:DNA, Satellite; D004279:DNA, Viral