title: Performance assessment of eight high-throughput PCR assays for viral load quantitation of oncogenic HPV types.
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.
authors.affiliation: Arizona Cancer Center, The University of Arizona, Tucson, USA.
authors.forename: Roberto
authors.initials: R
authors.lastname: Flores-Munguia
authors.forename: Erin
authors.initials: E
authors.lastname: Siegel
authors.forename: Walter T
authors.initials: WT
authors.lastname: Klimecki
authors.forename: Anna R
authors.initials: AR
authors.lastname: Giuliano
mesh_terms: D015344:DNA Probes, HPV; D004279:DNA, Viral / Q000032:analysis / Q000302:isolation & purification; D005260:Female; D006801:Humans; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D002583:Uterine Cervical Neoplasms / Q000821:virology*; D014626:Vaginal Smears; D019562:Viral Load; D002578:Uterine Cervical Dysplasia / Q000821:virology*
chemical_list: D015344:DNA Probes, HPV; D004279:DNA, Viral