Rapid detection of Epstein-Barr viral DNA by nonisotopic in situ hybridization. Correlation with the polymerase chain reaction.

Abstract

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.