title: Development of a sensitive and specific multiplex PCR method combined with DNA microarray primer extension to detect Betapapillomavirus types.
abstract: Emerging lines of evidence indicate that the cutaneous human papillomavirus (HPV) types that belong to the genus Betapapillomavirus (beta HPV) are involved in the development of nonmelanoma skin cancer. Unlike the situation for mucosal HPV types, highly sensitive and reliable methods to identify characterized cutaneous HPV types in a single assay are limited. Here, we describe a novel one-shot method for the detection of all characterized beta HPV types, namely, HPV type 5 (HPV5), 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96. This assay combines two different techniques: multiplex PCR using HPV type-specific primers for amplification of each E7 gene and array primer extension (APEX) for typing. This method has been validated using clinical samples which were analyzed simultaneously for the presence of cutaneous HPV types by two additional methods, i.e., the FAP59/64 PCR protocol and a commercially available PCR-reverse hybridization assay (PM-PCR RHA). Our data show good agreement between the results obtained with the multiplex PCR/APEX assay and the PM-PCR RHA method (overall HPV positivity of 92.2% for multiplex PCR/APEX assay versus 90.6% with the PM-PCR RHA) (kappa value, 50; 95% confidence interval, 13 to 88). In addition, the multiplex PCR/APEX assay showed higher sensitivity than the PM-PCR RHA did. This favorable feature and the high-throughput potential make this assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of cutaneous types in skin cancer.
authors.affiliation: International Agency for Research on Cancer, Lyon, France.
authors.forename: Tarik
authors.initials: T
authors.lastname: Gheit
authors.forename: Gaëlle
authors.initials: G
authors.lastname: Billoud
authors.forename: Maurits N C
authors.initials: MN
authors.lastname: de Koning
authors.forename: Federica
authors.initials: F
authors.lastname: Gemignani
authors.forename: Ola
authors.initials: O
authors.lastname: Forslund
authors.forename: Bakary S
authors.initials: BS
authors.lastname: Sylla
authors.forename: Salvatore
authors.initials: S
authors.lastname: Vaccarella
authors.forename: Silvia
authors.initials: S
authors.lastname: Franceschi
authors.forename: Stefano
authors.initials: S
authors.lastname: Landi
authors.forename: Wim G V
authors.initials: WG
authors.lastname: Quint
authors.forename: Federico
authors.initials: F
authors.lastname: Canzian
authors.forename: Massimo
authors.initials: M
authors.lastname: Tommasino
mesh_terms: D052686:Betapapillomavirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D005838:Genotype; D006801:Humans; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D050725:Papillomavirus E7 Proteins / Q000235:genetics; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity
chemical_list: D050725:Papillomavirus E7 Proteins