In situ hybridization : methodology and application to the diagnosis of certain viral infections

Abstract

In situ hybridization has become an increasingly popular method of detecting viral DNA or RNA in tissue sections. The major advantage of the technique over other molecular biology protocols, such as Southern blotting, is that the target nucleic acids can be very precisely identified within cells which retain their characteristic histopathological features. A further advantage is that fixed as well as fresh tissue can be used. This makes in situ hybridization ideal for retrospective studies and for the diagnosis of infections where only limited amounts of tissue arc available. We are currently using in situ techniques to investigate the epidemiology and cell biology of human papillomavirus (HPV)-asscciatcd cervical dysplasia and cancer, and molluscum contagiosum infections. Several different protocols, two using radioactive DNA probes and one involving the use of digoxigenin DNA probes, have been compared in terms of sensitivity and specificity. Nonradioactive probes may be preferred if speed and safety for laboratory personnel are more important than optimal sensitivity. As part of a retrospective study into the association of HPV with cervical cancer biopsies, we have compared results obtained by in situ hybridization with those obtained by Southern or dot blots on fresh tissue from the period 1986-7, using ³²P-labelled HPV 6/11, 16 and 18 DNA probes. In general, the results were in agreement: no cancers were found to contain HPV 6/11 only, the majority contained HPV 16 only, while a small number were positive for both HPV 6/11 and 16. However, the in situ method produced a larger percentage of negative results. This outcome is not surprising since the sensitivity of in situ hybridization, even under optimal conditions, is unlikely to reach that achieved by conventional probing on fresh tissue.