title: A new PCR-based assay amplifies the E6-E7 genes of most mucosal human papillomaviruses (HPV).
abstract: We established a new assay to detect the E6-E7 DNA of mucosal human papillomaviruses (HPV) by a PCR-based method using four pairs of degenerate LCR and E7 primers (LCR-E7 PCR). This assay amplifies the full length of E6 and the N-terminal part of E7. HPV typing was performed using restriction-fragment-length polymorphism (RFLP), and by analyzing the sequences of cloned PCR products. We compared this assay with the first generation hybrid captured assay (HCA-I) and the MY09/11-PCR method. LCR-E7 PCR was able to detect more than 34 mucosal HPV types and theoretically should detect two additional types. LCR-157 PCR and HCA-I detected HPV DNA in 70% (69/99) and 55% (54/99) of low-grade cervical intraepithelial lesions (LSIL), 89% (105/118) and 76% (90/118) of high-grade cervical intraepithelial lesions (HSIL), and 90% (56/62) and 79% (49/62) of invasive squamous cell carcinomas (SCC), respectively. LCR-E7 PCR was more sensitive than the HCA-1 test. Discordant results between the LCR-E7 and MY 11/09-PCR tests were observed in one of 185 (0.5%) normal samples, seven of 85 (8.2%) LSIL samples, seven of 82 (8.5%) HSIL samples, and four of 72 (5.6%) SCC samples. The discordant results were mostly observed in samples with a low-copy number of the HPV genome or with multiple HPV infection. The sensitivity of LCR-E7 PCR was equivalent to that of MY 11/09 ECR, and false positives were less frequent in LCR-E7 PCR. LCR-E7 PCR may be useful for determining the biological activity of detected HPV types, since this method amplifies the entire E6 gene.
authors.affiliation: School of Health Sciences, Kanazawa University, Ishikawa, Japan. tsasa@med.kanazawa-u.ac.jp
authors.forename: T
authors.initials: T
authors.lastname: Sasagawa
authors.forename: Y
authors.initials: Y
authors.lastname: Minemoto
authors.forename: W
authors.initials: W
authors.lastname: Basha
authors.forename: H
authors.initials: H
authors.lastname: Yamazaki
authors.forename: M
authors.initials: M
authors.lastname: Nakamura
authors.forename: H
authors.initials: H
authors.lastname: Yoshimoto
authors.forename: J
authors.initials: J
authors.lastname: Sakaike
authors.forename: M
authors.initials: M
authors.lastname: Inoue
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D000595:Amino Acid Sequence; D036022:Capsid Proteins; D002294:Carcinoma, Squamous Cell / Q000821:virology; D015331:Cohort Studies; D017931:DNA Primers / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics; D005260:Female; D006801:Humans; D007564:Japan; D008875:Middle Aged; D008969:Molecular Sequence Data; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D030361:Papillomavirus Infections / Q000821:virology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000821:virology; D002583:Uterine Cervical Neoplasms / Q000821:virology
chemical_list: D036022:Capsid Proteins; D017931:DNA Primers; D004279:DNA, Viral; C114580:HPV L1 protein, Human papillomavirus; D009856:Oncogene Proteins, Viral