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Journal of Clinical Microbiology, April 2008, p. 1161-1168, Vol. 46, No. 4
0095-1137/08/$08.00+0     doi:10.1128/JCM.00793-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Genotyping of Human Papillomaviruses by a Novel One-Step Typing Method with Multiplex PCR and Clinical Applications

GLab Pathology Center Co., Ltd., Sapporo, Japan,¹ Department of Gastroenterology and Hematology, Graduate School of Medicine, Hokkaido University, Sapporo, Japan,² EVE Women's Clinic, Sapporo, Japan,³ Department of Medical Management and Informatics, Hokkaido Information University, Sapporo, Japan,⁴ GeneticLab Co., Ltd., Sapporo, Japan⁵

Received 13 April 2007/ Returned for modification 11 June 2007/ Accepted 21 January 2008

We describe here a rapid, high-throughput genotyping procedure that allows the simultaneous detection of 16 high- and low-risk genital human papillomavirus (HPV) types by multiplex PCR in a single reaction tube. Multiplex PCR is based on the amplification of HPV DNA by sets of HPV genotype-specific primers, and the genotypes of HPV are visually identified by the sizes of amplicons after they are separated by capillary electrophoresis. The procedure does not include a hybridization step with HPV-specific probes and is rapid and labor-saving. We detected all 16 HPV genotypes (types 16, 58, 52, 51, 56, 31, 18, 39, 66, 59, 6, 33, 30, 35, 45, and 11) with a high sensitivity and a high degree of reproducibility. By using this newly developed method, we conducted a pilot study to examine the correlation between the prevalence and genotype distributions of HPV and the cytological group classifications for 547 cervical samples. Compared with the group of samples considered normal (14.7%), there was a significant increase in the prevalence of HPV in women with atypical squamous cells of unknown significance (61.3%), low-grade intraepithelial lesions (75.8%), and high-grade intraepithelial lesions (HSILs) (82.2%). The prevalence and distribution of type 58 were correlated with cytological malignancies, with the highest prevalence in women with HSILs. In conclusion, the novel multiplex PCR method described appears to be highly suitable not only for the screening of cervical cancer precursor lesions but also for the characterization of genotype distributions in large-scale epidemiological studies and HPV vaccination trials.

Published ahead of print on 30 January 2008.