title: Human papillomavirus typing by single tube multiplex amplification in real time (SMART): the PapillomaFinder® SMART 20 assay.
abstract: BACKGROUND
  High-risk (hr) human papillomavirus (HPV) infections play a causal role in the development of cervical cancer. The detection of hrHPV is, therefore, advocated in cervical cancer screening programs.
  
  
  OBJECTIVE
  The aim of this study was to determine the performance of a novel HPV typing assay, PapillomaFinder® SMART 20. This is a one-tube-per-sample method, to be performed on standard real-time PCR platforms, using melting curve analysis to distinguish targets. The assay detects all 14 hrHPV types, of which 16, 18, 31, 33, 35, 39, 45, 52, 56 and 58 individually. HrHPV types 51, 59, 66 and 68 are detected in an hrHPV pool, and low-risk (lr) HPV types 6, 11, 40, 42, 43 and 44 in an lrHPV pool.
  
  
  METHODS
  The method was tested on HPV plasmid models, WHO and QCMD proficiency panels and a series of clinical cytological samples (n=45), the latter in comparison with a clinically validated real-time quantitative PCR.
  
  
  RESULTS
  Type-specificity of the test was 100% using plasmids, the WHO and QCMD panels. Sensitivity for hrHPV in single infections was 100% using the WHO and QCMD panels and cytological samples, with an analytical sensitivity of 10-25 copies per reaction for all HPV types tested. Of the 34 HPV types present in the 8 multiple infections in the WHO panel, 30 were detected. In all cytological samples at least one hrHPV type was found, in concordance with the clinically validated method. Only when the viral load of the dominant HPV types in multiple infections greatly exceeded that of the other types in the infection, those other types were not always detected.
  
  
  CONCLUSIONS
  PapillomaFinder® SMART 20 is a rapid, easy to perform, single tube HPV typing assay. The assay detects the 14 hrHPV types, and the 6 most important lrHPV types with a high sensitivity and type-specificity.
authors.affiliation: PathoFinder BV, Maastricht, The Netherlands.
authors.forename: J
authors.initials: J
authors.lastname: Ossel
authors.affiliation: Department of Obstetrics and Gynaecology, Maastricht University Medical Centre, Maastricht, The Netherlands; Department of Molecular Cell Biology, GROW - School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands.
authors.forename: R J
authors.initials: RJ
authors.lastname: Litjens
authors.affiliation: PathoFinder BV, Maastricht, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Reijans
authors.affiliation: PathoFinder BV, Maastricht, The Netherlands.
authors.forename: A A T P
authors.initials: AA
authors.lastname: Brink
authors.affiliation: Department of Molecular Cell Biology, GROW - School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: Ummelen
authors.affiliation: Department of Molecular Cell Biology, GROW - School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands.
authors.forename: F C
authors.initials: FC
authors.lastname: Ramaekers
authors.affiliation: Department of Molecular Cell Biology, GROW - School for Oncology and Developmental Biology, Maastricht University Medical Centre, Maastricht, The Netherlands.
authors.forename: A H N
authors.initials: AH
authors.lastname: Hopman
authors.affiliation: PathoFinder BV, Maastricht, The Netherlands. Electronic address: Guus.Simons@Pathofinder.com.
authors.forename: G
authors.initials: G
authors.lastname: Simons
mesh_terms: D005260:Female; D060005:Genotyping Techniques / Q000379:methods*; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D027383:Papillomaviridae / Q000145:classification* / Q000235:genetics*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D013997:Time Factors
keywords: Cervical cancer; Human papillomavirus; Multiplex; Real-time polymerase chain reaction; Typing