BACKGROUND: Cancer of the cervix is still a deadly disease. Since the finding of an association between human papillomavirus (HPV) and cervical carcinoma, the development of a reliable means of detecting viral DNA in cervical scrapes has become a priority. We have used the polymerase chain reaction to detect DNA from HPV types 16 and 18 in cervical scrapes. EXPERIMENTAL DESIGN: We designed a protocol that minimizes manipulation steps and improves control of the reaction. Our technique involves elaboration of a unique reaction mixture (core reagent) containing all reagents except Taq polymerase. Each cervical sample from controls and patients treated during the same experiment, received an aliquot of this core reagent, with the DNA polymerase added just before dispensing. The results of the amplification are visualized on a polyacrylamide gel stained with ethidium bromide. Positive results for viral DNA are confirmed by restriction mapping of the amplified products. We used HeLa cells as the positive control for HPV 18 and negative control for HPV 16 and SiHa cells for the reciprocal controls. As an internal control, we used a target in the exon 3 of the human embryonic myosin heavy chain gene. RESULTS: The polymerase chain reaction in our experiments assure a sensitivity at least equal to two copies of target per cell. We analyzed 120 cervical smears with normal cytology; only 4% gave a positive result for HPV 16. We did not detect any HPV 18 DNA. CONCLUSIONS: This prevalence, which is among the lowest reported in the literature to date, supports the concept that HPV detection may have value in aiding the prevention of cervical cancer.
BACKGROUND:Cancer of the cervix is still a deadly disease. Since the finding of an association between humanpapillomavirus (HPV) and cervical carcinoma, the development of a reliable means of detecting viral DNA in cervical scrapes has become a priority. We have used the polymerase chain reaction to detect DNA from HPV types 16 and 18 in cervical scrapes. EXPERIMENTAL DESIGN: We designed a protocol that minimizes manipulation steps and improves control of the reaction. Our technique involves elaboration of a unique reaction mixture (core reagent) containing all reagents except Taq polymerase. Each cervical sample from controls and patients treated during the same experiment, received an aliquot of this core reagent, with the DNA polymerase added just before dispensing. The results of the amplification are visualized on a polyacrylamide gel stained with ethidium bromide. Positive results for viral DNA are confirmed by restriction mapping of the amplified products. We used HeLa cells as the positive control for HPV 18 and negative control for HPV 16 and SiHa cells for the reciprocal controls. As an internal control, we used a target in the exon 3 of the humanembryonic myosin heavy chain gene. RESULTS: The polymerase chain reaction in our experiments assure a sensitivity at least equal to two copies of target per cell. We analyzed 120 cervical smears with normal cytology; only 4% gave a positive result for HPV 16. We did not detect any HPV 18 DNA. CONCLUSIONS: This prevalence, which is among the lowest reported in the literature to date, supports the concept that HPV detection may have value in aiding the prevention of cervical cancer.
Authors: I Bourgault Villada; M Moyal Barracco; S Berville; M L Bafounta; C Longvert; V Prémel; P Villefroy; E Jullian; T Clerici; B Paniel; B Maillère; J Choppin; J G Guillet Journal: Clin Exp Immunol Date: 2009-07-30 Impact factor: 4.330