Analytical methods for evaluation on whole cells of human papillomavirus infection.
Analytical methods for evaluation on whole cells of human papillomavirus infection. Human papillomavirus (HPV) infection is currently identified by the presence of viral DNA using molecular biology. As in situ hybridization is valuable for HPV-DNA detection mainly with non-isotopic probes, we evaluated the sensitivity of various techniques, using as models three cell lines containing different copy numbers of HPV DNA/cell (CaSki with 600 copies of HPV 16, SiHa with 1-2 copies of HPV 16, HeLa with 10-50 copies of HPV 18). Epifluorescence microscopy and flow cytometry allowed detection of 600 copies in CaSki cells; in addition, cell fixation was found to influence the fluorescent intensity. Several procedures were assayed to increase the sensitivity of in situ hybridization. The use of biotinylated HPV-16 oligonucleotides as probes was not effective, because only CaSki cells were positive. After amplification of HPV-16 or -18 DNA sequences with polymerase chain reaction (PCR) on whole cells in suspension and hybridization with plasmid probes, fluorescent hybridization spots were found in CaSki and HeLa cells by both epifluorescence microscopy and flow cytometry. The various procedures applied for revelation of DNA-DNA hybrids (use of phycoerythrin or cyanine instead of fluorescein, Pinkel's 3-step amplified system of fluorescein) did not enhance the sensitivity of in situ hybridization. HPV DNA was very effectively detected by cell examination under a laser-scanning confocal microscope, since 1-2 copies of HPV 16 were observed in SiHa cells without previous PCR amplification. Thus, the efficacy of in situ hybridization for HPV detection may be conditioned by different factors. Laser-scanning microscopy represents an alternative to the use of PCR amplification. These techniques are potentially useful to study single genes.
Chardonnet
Y
Y
Inserm U346, hôpital E-Herriot, Lyon, France.
Lizard
G
G
Chignol
M C
MC
Schmitt
D
D
D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000302:isolation & purification; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D017403:In Situ Hybridization / Q000379:methods*; D017404:In Situ Hybridization, Fluorescence; D018613:Microscopy, Confocal; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000821:virology*
D015344:DNA Probes, HPV; D004279:DNA, Viral