Detection and Quantitation of Human Papillomavirus by Using the Fluorescent 5′ Exonuclease Assay

A method for the detection and quantitation of oncogenic human papillomavirus (HPV) was developed by using the fluorescent 5' exonuclease assay. The method is based on the amplification of a 180-bp fragment from the 3' part of the E1 open reading frame in a single PCR with type-specific probes for HPV types 16, 18, 31, 33, and 35. The probes can be used separately or in combinations of up to three probes per assay. Quantitation over a range of 10(1) to 10(6) initial HPV copies was possible by using real-time detection of the accumulation of fluorescence with cycle number. Reconstitution experiments, performed to mimic mixed infections, showed that individual HPV types can be detected down to a ratio of about 1% in a mixture. The performance of the assay depends on DNA quality, the presence of PCR inhibitors, and the number of different probes used simultaneously. This homogeneous assay provides a fast and sensitive way of screening for oncogenic HPV types in biopsy specimens as well as cervical smear samples. The closed-tube nature of the assay and the inclusion of uracil N'-glycosylase reduces cross contamination of PCR products to a minimum. A similar assay for beta-actin was used in parallel for quantitation of genomic DNA. After normalizing the samples for genomic DNA content, the mean number of HPV copies per cell could be calculated.