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Journal of Clinical Microbiology, August 2007, p. 2537-2544, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00747-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Development of a Sensitive and Specific Multiplex PCR Method Combined with DNA Microarray Primer Extension To Detect Betapapillomavirus Types{triangledown}

Tarik Gheit,1 Gaëlle Billoud,1 Maurits N. C. de Koning,2 Federica Gemignani,3 Ola Forslund,5 Bakary S. Sylla,1 Salvatore Vaccarella,1 Silvia Franceschi,1 Stefano Landi,3 Wim G. V. Quint,2 Federico Canzian,4 and Massimo Tommasino1*

International Agency for Research on Cancer, Lyon, France,1 DDL Diagnostic Laboratory, 2275 CX Voorburg, The Netherlands,2 Genetica, Dipartmento di Scienze Uomo e Ambiente, University of Pisa, Pisa, Italy,3 Deutsches Krebsforschungszentrum, D-69120 Heidelberg, Germany,4 Department of Medical Microbiology, Malmö University Hospital, Lund University, S-205 02 Malmö, Sweden5

Received 6 April 2007/ Returned for modification 12 May 2007/ Accepted 10 June 2007

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.

* Corresponding author. Mailing address: Infections and Cancer Biology Group, International Agency for Research on Cancer, 150, cours Albert-Thomas, 69372 Lyon, France. Phone: 33-4-72738191. Fax: 33-4-72738442. E-mail: tommasino{at}iarc.fr

{triangledown} Published ahead of print on 20 June 2007.

Journal of Clinical Microbiology, August 2007, p. 2537-2544, Vol. 45, No. 8
0095-1137/07/$08.00+0     doi:10.1128/JCM.00747-07
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Miller, M. B., Tang, Y.-W. (2009). Basic Concepts of Microarrays and Potential Applications in Clinical Microbiology. Clin. Microbiol. Rev. 22: 611-633 [Abstract] [Full Text]  


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