title: High-throughput detection, genotyping and quantification of the human papillomavirus using real-time PCR.
abstract: BACKGROUND
  The establishment of the causal relationship between high-risk human papillomavirus (HR-HPV) infection and cervical cancer and its precursors has resulted in the development of HPV DNA detection systems. Currently, real-time PCR assays for the detection of HPV, such as the RealTime High Risk (HR) HPV assay (Abbott) and the cobas® 4800 HPV Test (Roche Molecular Diagnostics) are commercially available. However, none of them enables the detection and typing of all HR-HPV types in a clinical high-throughput setting. This paper describes the laboratory workflow and the validation of a type-specific real-time quantitative PCR (qPCR) assay for high-throughput HPV detection, genotyping and quantification. This assay is routinely applied in a liquid-based cytology screening setting (700 samples in 24 h) and was used in many epidemiological and clinical studies.
  
  
  METHODS
  The TaqMan-based qPCR assay enables the detection of 17 HPV genotypes and β-globin in seven multiplex reactions. These HPV types include all 12 high-risk types (HPV16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59), three probably high-risk types (HPV53, 66 and 68), one low-risk type (HPV6) and one undetermined risk type (HPV67).
  
  
  RESULTS
  An analytical sensitivity of ≤100 copies was obtained for all the HPV types. The analytical specificity of each primer pair was 100% and an intra- and inter-run variability of <6.4% was observed.
  
  
  CONCLUSIONS
  The type-specific real-time PCR approach enables detection of 17 HPV types, identification of the HPV type and determination of the viral load in a single sensitive assay suitable for high-throughput screening.
authors.affiliation: Applied Molecular Biology Research (AMBIOR) Group, Laboratory of Cell Biology and Histology, University of Antwerp, Antwerp, Belgium. Isabel.Micalessi@ua.ac.be
authors.forename: Isabel M
authors.initials: IM
authors.lastname: Micalessi
authors.forename: Gaëlle A V
authors.initials: GA
authors.lastname: Boulet
authors.forename: Johannes J
authors.initials: JJ
authors.lastname: Bogers
authors.forename: Ina H
authors.initials: IH
authors.lastname: Benoy
authors.forename: Christophe E
authors.initials: CE
authors.lastname: Depuydt
mesh_terms: D004279:DNA, Viral / Q000032:analysis / Q000235:genetics; D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D027383:Papillomaviridae / Q000235:genetics* / Q000302:isolation & purification* / Q000502:physiology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D019562:Viral Load
chemical_list: D004279:DNA, Viral