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Development of a sensitive and specific multiplex PCR method combined with DNA microarray primer extension to detect beta-papillomavirus types.

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Author

  • Tarik Gheit
  • Gaelle Billoud
  • Maurits N. C. de Koning
  • Federica Gemignani
  • Ola Forslund
  • Bakary S. Sylla
  • Salvatore Vaccarella
  • Silvia Franceschi
  • Stefano Landi
  • Wim G. V. Quint
  • Federico Canzian
  • Massimo Tommasino
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Summary, in English

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.

Publishing year

2007

Language

English

Pages

2537-2544

Publication/Series

Journal of Clinical Microbiology

Volume

45

Issue

8

Document type

Journal article

Publisher

American Society for Microbiology

Topic

  • Microbiology in the medical area

Status

Published

Research group

  • Clinical Microbiology, Malmö

ISBN/ISSN/Other

  • ISSN: 1098-660X