A quantitative polymerase chain reaction-enzyme immunoassay for accurate measurements of human papillomavirus type 16 DNA levels in cervical scrapings

A quantitative polymerase chain reaction-enzyme immunoassay (Q-PCR-EIA) was developed to measure the amount of human papillomavirus (HPV) 16 DNA per g enome equivalent in cervical scrapings. The quantitative approach was based on a combined competitive PCR for both HPV 16, using the general primer GP 5+/6+ PCR, and beta-globin DNA, The two competitive PCRs involve co-amplifi cation of target sequences and exogenously added DNA constructs carrying a rearranged 30 bp sequence in the probe-binding region. The accuracy of quan tification by combining the two competitive PCR assays was validated on mix tures of HPV 16 containing cervical cancer cells of CaSki and SiHa cell lin es. Comparison of this fully quantitative PCR assay with two semi-quantitat ive HPV PCR assays on a series of crude cell suspensions from HPV 16 contai ning cervical scrapings revealed remarkable differences in the calculated r elative HPV load between samples. We found evidence that correction for bot h intertube variations in PCR efficiency and number of input cells/integrit y of DNA significantly influence the outcome of studies on viral DNA load i n crude cell suspensions of cervical scrapings. Therefore, accurate measure ments on viral DNA load in cervical scrapings require corrections for these phenomena, which can be achieved by application of this fully quantitative approach.