Abstract
Infection with mucosotropic human papillomavirus (HPV) is the necessary cause of cervical intraepithelial neoplasia. Several epidemiological studies suggest that HPV viral load can be a risk factor of cervical dysplasia. The purpose of the present study was to evaluate a methodology to determine HPV viral load of eight oncogenic HPV types (16, 18, 31, 39, 45, 51, 52, and 58). The quantitation assay is based on a high-throughput real-time PCR. The E6-E7 region of HPV types 16, 18, 45, and 51 were used to amplify specific DNA sequences and cloned into a plasmid vector. The constructs for HPV types 16, 18, 45, and 51, and the whole genome for HPV types 31, 39, 52, and 58 were quantitated using a limiting dilution analysis and used to create standard curves. Type-specific HPV clones were used to determine specificity, linearity, and intra- and inter-assay variation. The sensitivity of our assay covered a dynamic range of up to seven orders of magnitude with a coefficient of intra-assay variation less than 6% and the inter-assay variation less than 20%. No cross-reactivity was observed on any of the type-specific standard curves when phylogenetically close HPV types were used as templates. Our real-time PCR methodologies are highly reproducible, sensitive, and specific over a sevenfold magnitude dynamic range.