Visualization of single copies of the Epstein-Barr virus genome by in situ hybridization

Conditions have been established for demonstrating small numbers of genes of the Epstein-Barr virus (EBV) in B-lymphoid cells by in situ hybridization using biotinylated EBV-specific DNA from cloned BamHI fragments of the viral genome. Single copies of EBV genomes were successfully visualized with minimal background when the probe concentration was 0.2 micrograms/ml, the DNA denaturation step was performed at 100 degrees C, and the immunochemical detection system employed a three-layer peroxidase protocol with gold-silver amplification of the diaminobenzidine substrate. The minimal target DNA detectable was about 10 kilobase pairs. In the case of sectioned cells fixed overnight with formalin, simulating conditions used in routine tissue fixation, this approach failed to demonstrate EBV DNA present at less than 100 copies per cell, that is, at the level found in Raji cells. However, when denaturation was performed using microwave irradiation with the other optimized conditions maintained, EBV DNA could be visualized in 10-20% of such cells, although not in cells known to contain fewer than 10 copies per cell. Thus, microwave irradiation partially overcomes the limit of DNA target detection imposed by formalin.