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

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 intr...

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Published inJournal of Clinical Microbiology Vol. 44; no. 5; pp. 1792 - 1800
Main Authors Koning, Maurits de, Quint, Wim, Struijk, Linda, Kleter, Bernhard, Wanningen, Patrick, Doorn, Leen-Jan van, Weissenborn, Sönke Jan, Feltkamp, Mariet, Schegget, Jan ter
Format Journal Article
LanguageEnglish
Published Washington, DC American Society for Microbiology 01.05.2006
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Summary: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.
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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@ddl.nl.
ISSN:0095-1137
1098-660X
1098-5530
DOI:10.1128/JCM.44.5.1792-1800.2006