PROSPECTIVE METHODS FOR HUMAN PAPILLOMAVIRUS DETECTION BY FLUORESCENT IN-SITU HYBRIDIZATION AND LASER EXCITATION

As some human papillomaviruses (HPV) have an oncogenic potential, it is of interest to develop cytological methods which allow the detection of low copy numbers of HPV DNA. The human uterine carcinoma cell lines CaSki, SiHa and HeLa were used since they contain different copy numbers of HPV DNA per cell: 500-600 copies of HPV16, 1-2 copies of HPV 16 and 20-50 copies of HPV18, respectively. HPV DNA was revealed by fluorescent in situ hybridization (FISH) with biotinylated probes. The sensitivity of detection of viral DNA depends not only on the FISH reaction conditions but also on some characteristics of the flow cytometer (FC). By flow cytometry (FCM), the detection of viral DNA by FISH was limited to 500-600 copies per cell. The fluorescence signal was multiplied by a factor of 3 after successive fixations in 4% paraformaldehyde and 70% ethanol as compared to fixation in 70% ethanol. It was also enhanced after in situ amplification with polymerase chain reaction (PCR) so that 50 copies of HPV18 were detected in HeLa cells. The choice of FC was important since a stronger signal was obtained on FACScan than on FACStar plus. Furthermore, closed flow chamber gave better results than nozzle with jet in air, The detection of 1-2 copies of HPV DNA type 16 in SiHa cells was possible with laser scanning confocal microscopy (LSCM) without in situ PCR. Thus, LSCM may be more adapted than FCM to detect low copy numbers of HPV DNA.