Human papillomavirus detection by non isotopic in situ hybridization, in situ hybridization with signal amplification and in situ Polymerase Chain Reaction

Classical in situ hybridization (ISH) with biotinylated probes makes it pos sible to detect and localize human papillomavirus (HPV) nucleic acid sequen ces in cytological and histological materials. This method is however of li mited value in the detection of a few copies of the virus. Moreover the spe cificity of such a technique is not always convincing when ISH signals are small and/or of low intensity. Recently, much attention has been focused on the utility of the in vitro polymerase chain reaction (PCR) and especially on PCR-single strand conformation polymorphism (SSCP) to amplify small amo unts of viral DNA with accurate hybrid specificity. But the latter method r equires nucleic acid extraction and tissue destruction. Thus, correlation b etween the PCR results and histological findings is not possible. Hence, th e aim of our current study was to apply to HeLa cells and cervical formalin -fixed and paraffin-embedded biopsies, a novel procedure of ISH signal ampl ification, the catalyzed signal amplification (CSA). Such a procedure is ba sed on the deposition of streptavidin-horseradish peroxidase catalyzing the deposition of biotinylated tyramide molecules on the location of the probe d target. The biotin accumulation is then detected with streptavidin peroxi dase and diaminobenzidine. The results were compared with those obtained by direct and indirect in situ PCR. The catalysed signal amplification succes sfully increased the sensitivity and efficiency of ISH for the detection of rare sequences in HPV infected cells and histological materials. Such a me thod was found simpler and faster than in situ PCR and tissue morphology wa s better preserved.