Laser scanning confocal microscopy and quantitative microscopy with a charge coupled device camera improve detection of human papillomavirus DNA revealed by fluorescence in situ hybridization.
Epithelial cervical CaSki, SiHa and HeLa cells containing respectively 600 copies of human papillomavirus (HPV) DNA type 16, 1-2 copies of HPV DNA type 16 and 10-50 copies of HPV DNA type 18 were used as model to detect different quantities of integrated HPV genome. The HPV DNA was identified on cell deposits with specific biotinylated DNA probes either by enzymatic in situ hybridization (EISH) or fluorescence in situ hybridization (FISH) involving successively a rabbit anti-biotin antibody, a biotinylated goat anti-rabbit antibody and streptavidin-alkaline phosphatase complex or streptavidin-fluorescein isothiocyanate complex. With brightfield microscopy and EISH, hybridization spots were observed in CaSki and HeLa cells but hardly any in SiHa cells. With fluorescence microscopy and FISH, hybridization spots were clearly seen only on CaSki cell nuclei. In an attempt to improve the detection of low quantities of HPV DNA signals revealed by FISH, laser scanning confocal microscopy (LSCM) and quantitative microscopy with an intensified charge coupled device (CCD) camera were used. With both LSCM and quantitative microscopy, as few as 1-2 copies of HPV DNA were detected and found to be confined to cell nuclei counterstained with propidium iodide. Under Nomarski phase contrast, a good preservation of the cell structure was observed. With quantitative microscopy, differences in the number, size, total area and integrated fluorescence intensity of hybridization spots per nucleus were revealed between CaSki, SiHa and HeLa cells.(ABSTRACT TRUNCATED AT 250 WORDS)
Lizard
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G
Centre Commun de Cytométrie en Flux, INSERM U80, Hôpital E. Herriot, Lyon, France.
Chignol
M C
MC
Souchier
C
C
Schmitt
D
D
Chardonnet
Y
Y
D002467:Cell Nucleus / Q000737:chemistry / Q000821:virology; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D016679:Genome, Viral; D006367:HeLa Cells; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D007834:Lasers; D008853:Microscopy / Q000379:methods; D008856:Microscopy, Fluorescence; D027383:Papillomaviridae / Q000235:genetics*; D030361:Papillomavirus Infections / Q000235:genetics / Q000821:virology; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000235:genetics / Q000821:virology; D014594:Uterine Neoplasms / Q000821:virology; D016662:Virus Integration
D004279:DNA, Viral