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Journal of Clinical Microbiology, May 2006, p. 1792-1800, Vol. 44, No. 5
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.5.1792-1800.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Evaluation of a Novel Highly Sensitive, Broad-Spectrum PCR-Reverse Hybridization Assay for Detection and Identification of Beta-Papillomavirus DNA

Maurits de Koning,1,2 Wim Quint,1* Linda Struijk,2 Bernhard Kleter,1 Patrick Wanningen,2 Leen-Jan van Doorn,1 Sönke Jan Weissenborn,3 Mariet Feltkamp,2 and Jan ter Schegget1,2

Delft Diagnostic Laboratory, Voorburg, The Netherlands,1 Department of Medical Microbiology, Center of Infectious Diseases, Leiden University Medical Center, Leiden, The Netherlands,2 Institute of Virology, University of Cologne, Germany3

Received 5 October 2005/ Returned for modification 16 November 2005/ Accepted 15 February 2006

Human papillomavirus can be detected by amplification of viral DNA. A novel one-step PCR (PM-PCR) was evaluated for amplification of a 117-bp fragment from the E1 region. It permitted ultrasensitive detection of all 25 known human papillomavirus genotypes from the beta-papillomavirus genus. The intra- and intertypic sequence variations of the 77-bp interprimer region were studied. Genotype-specific probes as well as general probes were selected for the 25 established beta-papillomavirus types, and a reverse hybridization assay (RHA) was developed (PM-PCR RHA method). The analytical sensitivity of the PM-PCR RHA method was 10 to 100 viral genomes. The one-step PM-PCR turned out to be more sensitive than the previously described nested MaHa-PCR for beta-papillomavirus detection. The PM-PCR RHA method was able to detect and identify beta-papillomavirus types in frozen patient material as well as in poorly amplifiable material such as formalin-fixed, paraffin-embedded skin biopsy specimens. Inter- and intralaboratory variability experiments showed that the reproducibility of the assay was very high. In conclusion, the one-step PM-PCR together with the RHA allows extremely sensitive, specific, and reproducible detection of beta-papillomavirus DNA as well as reliable identification of beta-papillomavirus genotypes in both fresh and paraffin-embedded patient material.

* Corresponding author. Mailing address: Delft Diagnostic Laboratory, Fonteijnenburghlaan 5, 2275 CX Voorburg, The Netherlands. Phone: 31 703401670. Fax: 31 703401671. E-mail: w.g.v.quint{at}ddl.nl.

Journal of Clinical Microbiology, May 2006, p. 1792-1800, Vol. 44, No. 5
0095-1137/06/$08.00+0     doi:10.1128/JCM.44.5.1792-1800.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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