title: Genotyping of human papillomaviruses by a novel one-step typing method with multiplex PCR and clinical applications.
abstract: 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.
authors.affiliation: Department of Advanced Pathology Services, GLab Pathology Center Co., Ltd., 28-196, N9-W15, Chuo-ku, Sapporo 0600009, Japan. morie_nishiwaki@gene-lab.com
authors.forename: Morie
authors.initials: M
authors.lastname: Nishiwaki
authors.forename: Tomohiro
authors.initials: T
authors.lastname: Yamamoto
authors.forename: Somako
authors.initials: S
authors.lastname: Tone
authors.forename: Taichi
authors.initials: T
authors.lastname: Murai
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Ohkawara
authors.forename: Takakuni
authors.initials: T
authors.lastname: Matsunami
authors.forename: Motoiki
authors.initials: M
authors.lastname: Koizumi
authors.forename: Yoshitake
authors.initials: Y
authors.lastname: Takagi
authors.forename: Jun
authors.initials: J
authors.lastname: Yamaguchi
authors.forename: Nobuo
authors.initials: N
authors.lastname: Kondo
authors.forename: Jun
authors.initials: J
authors.lastname: Nishihira
authors.forename: Takeharu
authors.initials: T
authors.lastname: Horikawa
authors.forename: Takashi
authors.initials: T
authors.lastname: Yoshiki
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002584:Cervix Uteri / Q000821:virology*; D017931:DNA Primers; D019075:Electrophoresis, Capillary; D005260:Female; D005838:Genotype; D006801:Humans; D008875:Middle Aged; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity; D030361:Papillomavirus Infections / Q000453:epidemiology / Q000473:pathology / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D015995:Prevalence; D012680:Sensitivity and Specificity; D002583:Uterine Cervical Neoplasms / Q000453:epidemiology / Q000473:pathology / Q000821:virology; D002578:Uterine Cervical Dysplasia / Q000453:epidemiology / Q000473:pathology / Q000821:virology
chemical_list: D017931:DNA Primers