Detection of single copies of Epstein-Barr virus in paraffin wax sections by non-radioactive in situ hybridisation

A highly sensitive non-isotopic in situ hybridisation technique was developed for the localisation of Epstein-Barr virus (EBV) in paraffin wax embedded tissue sections. The method uses a repeated sequence of the EBV genome as a probe, labelled with the novel reporter molecule, digoxigenin. The method can identify individual copies of EBV by detection of both EBV DNA and highly localised RNA transcripts. A combination of careful proteolytic digestion of tissue sections, high temperature denaturation of probe and target DNA, and sensitive immunocytochemical detection are used to attain single copy sensitivity. The technique is quicker and simpler to perform than some other methods used for the identification of EBV, and provides simultaneous morphological information which cannot be obtained by methods using tissue extracts. This method permits the investigation of the role of EBV in neoplastic conditions of lymphoid and epithelial cells, and may prove valuable in determining the sites of latent virus in healthy subjects