A new PCR-based assay amplifies the E6-E7 genes of most mucosal human papillomaviruses (HPV)

Virus Res. 2000 Apr;67(2):127-39. doi: 10.1016/s0168-1702(00)00137-4.

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.

Publication types

  • Comparative Study

MeSH terms

  • Adolescent
  • Adult
  • Aged
  • Aged, 80 and over
  • Amino Acid Sequence
  • Capsid Proteins
  • Carcinoma, Squamous Cell / virology
  • Cohort Studies
  • DNA Primers / genetics
  • DNA, Viral / genetics
  • Female
  • Humans
  • Japan
  • Middle Aged
  • Molecular Sequence Data
  • Oncogene Proteins, Viral / genetics*
  • Papillomaviridae / classification
  • Papillomaviridae / genetics*
  • Papillomaviridae / isolation & purification
  • Papillomavirus Infections / virology
  • Polymerase Chain Reaction / methods*
  • Polymorphism, Restriction Fragment Length
  • Sensitivity and Specificity
  • Tumor Virus Infections / virology
  • Uterine Cervical Neoplasms / virology

Substances

  • Capsid Proteins
  • DNA Primers
  • DNA, Viral
  • HPV L1 protein, Human papillomavirus
  • Oncogene Proteins, Viral

Associated data

  • GENBANK/AF188643
  • GENBANK/AF188644
  • GENBANK/AF188645
  • GENBANK/AF188646
  • GENBANK/AF188647
  • GENBANK/AF188648
  • GENBANK/AF188649
  • GENBANK/AF188650
  • GENBANK/AF188651
  • GENBANK/AF188652
  • GENBANK/AF188653
  • GENBANK/AF188654
  • GENBANK/AF188655
  • GENBANK/AF188656
  • GENBANK/AF188657
  • GENBANK/AF188658
  • GENBANK/AF188659
  • GENBANK/AF188660
  • GENBANK/AF188661
  • GENBANK/AF188662
  • GENBANK/AF188663
  • GENBANK/AF188664
  • GENBANK/AF188665
  • GENBANK/AF188666
  • GENBANK/AF188667
  • GENBANK/AF188668
  • GENBANK/AF188669
  • GENBANK/AF188670
  • GENBANK/AF188671
  • GENBANK/AF188672