[Human papillomavirus typing by molecular hybridization in situ with biotinylated probes. Optimization of sample preparation and hybridization time].
In situ hybridization with non radioactive probes is more and more used to detect viral infections, especially human papillomavirus (HPV) infections. The quality of the reaction depends on several factors, such as sample preparation (including fixation and pretreatments). Their importance was evaluated on a model with cell lines including CaSki cells (harboring about 600 copies of HPV 16 DNA per cell) and Hela cells (containing 10-50 copies of HPV 18 DNA per cell). These cell lines were chosen in order to evaluate cytological and histological difficulties. Several parameters were studied: preparation of samples, fixation and hybridization duration. DNAs of biotinylated probes of HPV types 16 and 18 and cells were simultaneously denatured 10 min at 95 degrees C. Hybridization was carried out at 37 degrees C for various periods of time; it was followed by a 3-step reaction for detection of biotinylated DNA-DNA hybrids with immunoenzymatic staining using streptavidin-alkaline phosphatase complex. Typical intranuclear granulations were seen either in cell deposits fixed with acetone, methanol-acetic acid, paraformaldehyde, formaline, Bouin's, Baker's or Carnoy's fixatives; or in cytocentrifuged cells fixed with formaline, Bouin's or Baker's fixatives. The detergent pretreatment was unnecessary. On the contrary, the protease pretreatment was required with formaline, Bouin's or Baker's fixatives. In order to detect constantly HPV 16 in CaSki cells and HPV 18 in HeLa cells, hybridization should be performed for more than 4 h. The sensitivity of the technique could therefore be evaluated to few copies of HPV DNA per cell. This technique is reliable, sensitive and rapid; et can be applied to biopsy specimens fixed with Bouin's or Baker's fixatives and paraffin-embedded; it allows routine detection of HPV infections.
Guérin-Reverchon
I
I
INSERM U 209, CNRS URA 601, Hôpital E. Herriot, Lyon.
Chignol
M C
MC
Chardonnet
Y
Y
D001710:Biotin; D002460:Cell Line; D015342:DNA Probes; D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000032:analysis; D005404:Fixatives; D006367:HeLa Cells; D006801:Humans; D009693:Nucleic Acid Hybridization; D027383:Papillomaviridae / Q000145:classification*; D013997:Time Factors
D015342:DNA Probes; D015344:DNA Probes, HPV; D004279:DNA, Viral; D005404:Fixatives; D001710:Biotin
Typage de papillomavirus humains par hybridation moléculaire in situ avec des sondes biotinylées. Optimisation de la préparation des échantillons et du temps d'hybridation.