Rapid detection of Epstein-Barr viral DNA by nonisotopic in situ hybridization. Correlation with the polymerase chain reaction.
A nonisotopic, in situ hybridization procedure is described for detecting Epstein-Barr viral (EBV) DNA in cytopreparations of pepsin-digested, unfixed cells in less than 3 hours. The procedure, which uses an EBV Bam HI W DNA probe conjugated directly to horseradish peroxidase, first was optimized using an EBV-infected cell line (ARH-77) and then validated on four positive control specimens and six negative control specimens. Optimization studies demonstrated distinct nuclear hybridization signal in less than 10% of formaldehyde-fixed, pepsin-digested ARH-77 cells. When formaldehyde fixation was omitted, more than 50% of the pepsin-digested ARH-77 cells had strong nuclear staining. Unfixed cells from the four positive controls had detectable hybridization signal; the presence of EBV-DNA in these specimens also was confirmed by a polymerase chain reaction. There was no hybridization signal in the six negative control specimens. The results indicate that this nonisotopic, in situ hybridization procedure rapidly detects the presence of EBV-DNA in cytopreparations of unfixed, pepsin-digested cells from clinical specimens. There is a tradeoff, however, between assay sensitivity and preservation of cytologic details.
Samoszuk
M
M
Pathology Department, University of California, Irvine 92717.
D001402:B-Lymphocytes / Q000382:microbiology; D002460:Cell Line; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006689:Hodgkin Disease / Q000382:microbiology*; D006735:Horseradish Peroxidase; D006801:Humans; D007124:Immunoenzyme Techniques; D007244:Infectious Mononucleosis / Q000382:microbiology*; D009693:Nucleic Acid Hybridization; D016133:Polymerase Chain Reaction; D011237:Predictive Value of Tests
D015342:DNA Probes; D004279:DNA, Viral; D006735:Horseradish Peroxidase