title: Human papillomavirus detection by non isotopic in situ hybridization, in situ hybridization with signal amplification and in situ polymerase chain reaction.
abstract: Classical in situ hybridization (ISH) with biotinylated probes makes it possible to detect and localize human papillomavirus (HPV) nucleic acid sequences in cytological and histological materials. This method is however of limited value in the detection of a few copies of the virus. Moreover the specificity 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 amounts of viral DNA with accurate hybrid specificity. But the latter method requires nucleic acid extraction and tissue destruction. Thus, correlation between the PCR results and histological findings is not possible. Hence, the 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 amplification, the catalyzed signal amplification (CSA). Such a procedure is based on the deposition of streptavidin-horseradish peroxidase catalyzing the deposition of biotinylated tyramide molecules on the location of the probed target. The biotin accumulation is then detected with streptavidin peroxidase and diaminobenzidine. The results were compared with those obtained by direct and indirect in situ PCR. The catalysed signal amplification successfully increased the sensitivity and efficiency of ISH for the detection of rare sequences in HPV infected cells and histological materials. Such a method was found simpler and faster than in situ PCR and tissue morphology was better preserved.
authors.affiliation: Laboratoire de Virologie-Biologie Cellulaire-Immunologie, Faculté de Médecine et de Pharmacie, Besançon, France.
authors.forename: D
authors.initials: D
authors.lastname: Bettinger
authors.forename: B
authors.initials: B
authors.lastname: Bernard
authors.forename: D
authors.initials: D
authors.lastname: Riethmuller
authors.forename: I
authors.initials: I
authors.lastname: Greslin
authors.forename: C
authors.initials: C
authors.lastname: Gay
authors.forename: M
authors.initials: M
authors.lastname: Lab
authors.forename: E
authors.initials: E
authors.lastname: Seillès
authors.forename: C
authors.initials: C
authors.lastname: Mougin
mesh_terms: D001710:Biotin / Q000031:analogs & derivatives; D005260:Female; D006367:HeLa Cells / Q000821:virology; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D027383:Papillomaviridae / Q000235:genetics* / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D014439:Tyramine / Q000031:analogs & derivatives; D002578:Uterine Cervical Dysplasia / Q000473:pathology / Q000821:virology
chemical_list: C111551:biotinyltyramide; D001710:Biotin; D014439:Tyramine