Abstract
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.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bein G, Bitsch A, Hoyer J, Kirchner H (1991) The detection of human cytomegalovirus immediate-early antigen in peripheral blood leukocytes. J Immunol Methods 137:175–180
Dankner WM, McCutchan JA, Richman DD, Hirata K, Spector SA (1990) Localization of human cytomegalovirus in peripheral blood leukocytes by in situ hybridization. J Infect Dis 161:31–36
Fleckenstein B, Müller I, Collins J (1982) Cloning the complete human cytomegalovirus genome in cosmids. Gene 18:39–46
Ho M (1991) Observations from transplantation contributing to the understanding of pathogenesis of CMV infection. Transplant Proc 23 [Suppl 3]:104–109
Komminoth P (1992) Digoxigenin as an alternative probe labeling for in situ hybridization. Diagn Mol Pathol 1:142–150
Komminoth P, Merk FB, Leav I, Wolfe HJ, Roth J (1992a) Comparison of35S and digoxigenin-labeled RNA and oligonucleotide probes for in situ hybridization-expression of messenger RNA of the seminal vesicle secretion protein-II and androgen receptor genes in the rat prostate. Histochemistry 98:217–228
Komminoth P, Long AA, Ray R, Wolf HJ (1992b) In situ polymerase chain reaction detection of viral DNA, single copy genes and gene rearrangements in cell suspensions and cytospins. Diagn Mol Pathol 1:85–97
Komminoth P, Long AA (1993) In-situ polymerase chain reaction. An overview of methods, applications and limitations of a new molecular technique. Virchows Arch 64:67–73
Kuwano A, Ledbetter SA, Dobyns WB, Emanuel BS, Ledbetter DH (1991) Detection of deletions and cryptic translocations in Miller-Dieker syndrome by in situ hybridization. Am J Hum Genet 49:707–714
Lawrence JB, Villnave CA, Singer RH (1988) Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell 52:51–61
Lichter P, Ward DC (1990) Is non-isotopic in situ hybridization finally coming of age? Nature 345:93–94
Long AA, Komminoth P, Lee E, Wolfe HF (1993) Comparison of indirect and direct in situ polymerase chain reaction in cell preparations and tissue sections. Detection of viral DNA, gene rearrangements and chromosomal translocations. Histochemistry 99:151–162
McNeil JA, Johnson CV, Carter KC, Singer RH, Lawrence JB (1991) Localizing DNA and RNA within nuclei and chromosomes by fluorescence in situ hybridization. Genet Anat Tech Appl 8:41–58
Moorhead PS, Nowell PC, Mellman WJ, Battips DM, Hungerford DA (1960) Chromosome preparations of leukocytes cultured from human peripheral blood. Exp Cell Res 20:613–616
Peters BS, Beck EJ, Anderson S, Coleman C, Coker R, Main J, Migdal C, Harris JR, Pinching AJ (1991) Cytomegalovirus infection in AIDS. Patterns of disease, response to therapy and trends in survival. J Infect 23:129–137
Preiksaitis JK, Janowska-Wieczorek A (1991) Persistence of cytomegalovirus in human long-term bone marrow culture: relationship to hematopoiesis. J Med Virol 35:76–84
Raap AK, Rijke FM van de, Dirks RW, Sol CJ, Boom R, Ploeg M van der (1991) Bicolor fluorescence in situ hybridization to intron and exon mRNA sequences. Exp Cell Res 197:319–322
Rice GP, Schrier RD, Oldstone MB (1984) Cytomegalovirus infects human lymphocytes and monocytes: virus expression is restricted to immediate early gene products. Proc Natl Acad Sci USA 81:6134–6138
Riddell SR, Watanabe KS, Goodrich JM, Li CR, Agha ME, Greenberg PD (1992) Restoration of viral immunity in immunodeficient humans by the adoptive transfer of T cell clones. Science 257:238–241
Saltzman RL, Quirk MR, Jordan MC (1992) High levels of circulating cytomegalovirus DNA reflect visceral organ disease in viremic immunosuppressed patients other than marrow recipients. J Clin Invest 90:1832–1838
Schrier RD, Nelson JA, Oldstone MB (1985) Detection of human cytomegalovirus in peripheral blood lymphocytes in a natural infection. Science 230:1048–1051
Turtinen LW, Saltzman R, Jordan MC, Haase AT (1987) Interactions of human cytomegalovirus with leukocytes in vivo: analysis by in situ hybridization. Microb Pathog 3:287–297
Vernole P (1990) Digoxigenin-labeled probes can detect singlecopy genes in human metaphase chromosomes. Biotechniques 9:200–204
Wang HS, Riddell DC, Donald LJ, Cameron EC, Tonogai J, Holden JJA, Higgins M, Stromas I, White BN, Hamerton JL (1984) Mapping of a repetitive 1.8 kbKpnI sequence to the short arm of chromosome 15. Cytogenet Cell Genet 37:601–602
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hackstein, H., Jahn, G., Kirchner, H. et al. Fluorescence in situ hybridization with cosmid clones for the detection of human cytomegalovirus DNA in peripheral blood leukocytes. Histochem Cell Biol 106, 229–234 (1996). https://doi.org/10.1007/BF02484405
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF02484405