PCR for the detection of cervical HPV16 infection: The need for standardization

Early reports suggested an important role for the polymerase chain reaction (PCR) in epidemiological studies investigating the association between human pupillomavirus (HPV) and cervical neoplasia (Young et al., 1989; Editorial, 1989). Concern has been recently expressed about the reproducibility of this technique following reports of widely different estimates of the prevalence of HPV16 DNA sequences in normal and abnormal cervical epithelia (Frunco. 1991; Young et al. 1992). The possibility that these interstudy variations may reflect the use o f different primer sets has not preiiously been explored. Thus we have investigated how estimates of the prevalence of HPV16 in cytological material v a y with the choice ofprimer set used. Cervical smears were taken ,from 2 groups of women; those consulting a general practitioner for routine cervical screening and those referred to a colposcopy clinic for evaluation of cytological abnormalities. Two smear samples were taken from each patient; one for cytological examination, the other for PCR analysis. Cytological examination revealed no abnormality in 57 cases and the presence of at least dyskaryotic cells in 56 cases. Smear cells, collected into cold PBS, were pelleted and treated with 500 pl of 1 x PCR buffer (10 mM Tris-HCI, 1.5 mM MgCl2, 50 mM KCI, 0.1 mglml gelatine, p H 8.3) containing 100 pglinl proteinase K and 0.5% Tween 80 at 55°C for 60 min, then incubated at 94°C for 10 min. A 10-pl aliquot of this preparation was then amplified in a 100-pl reaction; amplification conditions and the detection of amplified products were as previously described (Yao et al., 1991). The specificity of all amplified products was confirmed by hybridization with the appropriate internal oligonucleotide probe and, where the L1 consensus primers were used, the type specificity of the umplifiedproduct was confirmed by hybridization with an HPV16-specific internal probe (see Table I ) . Five different sets of PCR primers were used to detect HPV16 in cervical smear samples. These primers target either the “late” region (LI consensus, LI specific) or the “early” region of the HPVl6 genome (Table I). Estimates of the prevalence of HPV16 in abnormal epithelium were similar, rangingfvom 38 to 43%. However, the prevalence of HPVI6 in normal epithelium ranged from 2 to 19% according to the primers used. With normal cervical smears, the primers recognizing the LI region of the viral genome produced identical estimates which were substantially in excess of those obtained using any of the remaining primer sets. That these differences were not attributable to differing detection sensitivities of the individual primers was established by using each of the primer sets in PCR analysis of decreasing concentrations of DNA from the HPVlb-positive CaSki cell line. Thus, whilst the L l consensus primers and the E7primers give a 10-fold difference in the detection of HPVI6 in normal smears, both primer sets have similar sensitivities, detecting as little as 50 fg of CaSki DNA equivalent to approximately 3.5 copies of the HPV16 genome. Taking both the normal and abnormal smears together (n = 115), a total of 52 samples were HPVl6-positive with one or more primer sets. Primers targeting the Ll region of the HPV16 genome detected 43 (83%) of all smears which were positive with at least one of the sets tested, whereas primers targeting the early region only detected an additional 9 (1 7%) positive cases. Varying the concentration of MgCl2 in the PCR did not alter the HPV16 detection rate with any of the primers used in this study. Our findings suggest that the prevalence of HPV16 DNA in cytological material varies with the primer set used and with the presence or absence of epithelial abnormality. The greatest difference between normal and abnormal material was in the prevalence of HPV16 DNA sequences . spec@ for the E6IE71 URR region. Whilst the overall prevalence of HPV16 in cytolog-