Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1995 Jul;33(7):1822–1828. doi: 10.1128/jcm.33.7.1822-1828.1995

Detection of genital human papillomavirus by single-tube nested PCR and type-specific oligonucleotide hybridization.

N Ylitalo 1, T Bergström 1, U Gyllensten 1
PMCID: PMC228277  PMID: 7665652

Abstract

Cervical cancer is, on a global scale, the second most common form of cancer in women. Development of cervical carcinoma is strongly associated with infection by certain types of human papillomavirus (HPV). To facilitate the detection and molecular typing of HPV in clinical samples, nested-PCR amplification systems were developed for regions of the E1 and L1 genes. The nested amplifications were performed in a single reaction tube, and shifting between inner and outer primer pairs was achieved by a two-phase amplification with different annealing temperatures. This method eliminates cross-contamination between samples during transfer from the first to the second amplification step. A set of type-specific oligonucleotide probes were designed for the E1 system and used to distinguish 19 genital HPV types. The sensitivities of our amplification systems compare favorably with that for the L1 system on the basis of the MY09-MY11 primer pair (M.M. Manos, Y. Ting, D. K. Wright, A. J. Lewis, T. R. Broker, and S. M. Wolinsky, Cancer Cells 7:209-214, 1989) and our systems can be used on materials such as HPV-infected cell lines, cytobrush samples, cancer biopsies, and recent as well as archival Papanicolaou (Pap) smears. The high sensitivity coupled with the effective elimination of contamination in the transfer between the two amplification steps of the nested PCR makes these systems suitable for research as well as clinical analyses.

Full Text

The Full Text of this article is available as a PDF (362.0 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Apple R. J., Erlich H. A., Klitz W., Manos M. M., Becker T. M., Wheeler C. M. HLA DR-DQ associations with cervical carcinoma show papillomavirus-type specificity. Nat Genet. 1994 Feb;6(2):157–162. doi: 10.1038/ng0294-157. [DOI] [PubMed] [Google Scholar]
  2. Bauer H. M., Ting Y., Greer C. E., Chambers J. C., Tashiro C. J., Chimera J., Reingold A., Manos M. M. Genital human papillomavirus infection in female university students as determined by a PCR-based method. JAMA. 1991 Jan 23;265(4):472–477. [PubMed] [Google Scholar]
  3. Campione-Piccardo J., Montpetit M. L., Grégoire L., Arella M. A highly conserved nucleotide string shared by all genomes of human papillomaviruses. Virus Genes. 1991 Oct;5(4):349–357. doi: 10.1007/BF00271533. [DOI] [PubMed] [Google Scholar]
  4. Evander M., Edlund K., Bodén E., Gustafsson A., Jonsson M., Karlsson R., Rylander E., Wadell G. Comparison of a one-step and a two-step polymerase chain reaction with degenerate general primers in a population-based study of human papillomavirus infection in young Swedish women. J Clin Microbiol. 1992 Apr;30(4):987–992. doi: 10.1128/jcm.30.4.987-992.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Evander M., Wadell G. A general primer pair for amplification and detection of genital human papillomavirus types. J Virol Methods. 1991 Feb-Mar;31(2-3):239–250. doi: 10.1016/0166-0934(91)90162-s. [DOI] [PubMed] [Google Scholar]
  6. Gall K., Pavicić D., Pavelić J., Audy-Jurković S., Pavelić K. PCR amplification of DNA from stained cytological smears. J Clin Pathol. 1993 Apr;46(4):378–379. doi: 10.1136/jcp.46.4.378. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Gustafsson L., Adami H. O. Natural history of cervical neoplasia: consistent results obtained by an identification technique. Br J Cancer. 1989 Jul;60(1):132–141. doi: 10.1038/bjc.1989.236. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gustafsson L., Adami H. O. Optimization of cervical cancer screening. Cancer Causes Control. 1992 Mar;3(2):125–136. doi: 10.1007/BF00051652. [DOI] [PubMed] [Google Scholar]
  9. Muñoz N., Bosch F. X. HPV and cervical neoplasia: review of case-control and cohort studies. IARC Sci Publ. 1992;(119):251–261. [PubMed] [Google Scholar]
  10. Rakoczy P., Sterrett G., Kulski J., Whitaker D., Hutchinson L., MacKenzie J., Pixley E. Time trends in the prevalence of human papillomavirus infections in archival Papanicolaou smears: analysis by cytology, DNA hybridization, and polymerase chain reaction. J Med Virol. 1990 Sep;32(1):10–17. doi: 10.1002/jmv.1890320103. [DOI] [PubMed] [Google Scholar]
  11. Scheffner M., Münger K., Byrne J. C., Howley P. M. The state of the p53 and retinoblastoma genes in human cervical carcinoma cell lines. Proc Natl Acad Sci U S A. 1991 Jul 1;88(13):5523–5527. doi: 10.1073/pnas.88.13.5523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Schiffman M. H., Bauer H. M., Hoover R. N., Glass A. G., Cadell D. M., Rush B. B., Scott D. R., Sherman M. E., Kurman R. J., Wacholder S. Epidemiologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia. J Natl Cancer Inst. 1993 Jun 16;85(12):958–964. doi: 10.1093/jnci/85.12.958. [DOI] [PubMed] [Google Scholar]
  13. Schiffman M. H., Bauer H. M., Lorincz A. T., Manos M. M., Byrne J. C., Glass A. G., Cadell D. M., Howley P. M. Comparison of Southern blot hybridization and polymerase chain reaction methods for the detection of human papillomavirus DNA. J Clin Microbiol. 1991 Mar;29(3):573–577. doi: 10.1128/jcm.29.3.573-577.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Smits H. L., Tieben L. M., Tjong-A-Hung S. P., Jebbink M. F., Minnaar R. P., Jansen C. L., ter Schegget J. Detection and typing of human papillomaviruses present in fixed and stained archival cervical smears by a consensus polymerase chain reaction and direct sequence analysis allow the identification of a broad spectrum of human papillomavirus types. J Gen Virol. 1992 Dec;73(Pt 12):3263–3268. doi: 10.1099/0022-1317-73-12-3263. [DOI] [PubMed] [Google Scholar]
  15. Snijders P. J., van den Brule A. J., Schrijnemakers H. F., Snow G., Meijer C. J., Walboomers J. M. The use of general primers in the polymerase chain reaction permits the detection of a broad spectrum of human papillomavirus genotypes. J Gen Virol. 1990 Jan;71(Pt 1):173–181. doi: 10.1099/0022-1317-71-1-173. [DOI] [PubMed] [Google Scholar]
  16. Syrjänen K., Mäntyjärvi R., Väyrynen M., Syrjänen S., Parkkinen S., Yliskoski M., Saarikoski S., Castrén O. Human papillomavirus (HPV) infections involved in the neoplastic process of the uterine cervix as established by prospective follow-up of 513 women for two years. Eur J Gynaecol Oncol. 1987;8(1):5–16. [PubMed] [Google Scholar]
  17. van den Brule A. J., Meijer C. J., Bakels V., Kenemans P., Walboomers J. M. Rapid detection of human papillomavirus in cervical scrapes by combined general primer-mediated and type-specific polymerase chain reaction. J Clin Microbiol. 1990 Dec;28(12):2739–2743. doi: 10.1128/jcm.28.12.2739-2743.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. van den Brule A. J., Snijders P. J., Gordijn R. L., Bleker O. P., Meijer C. J., Walboomers J. M. General primer-mediated polymerase chain reaction permits the detection of sequenced and still unsequenced human papillomavirus genotypes in cervical scrapes and carcinomas. Int J Cancer. 1990 Apr 15;45(4):644–649. doi: 10.1002/ijc.2910450412. [DOI] [PubMed] [Google Scholar]
  19. van den Brule A. J., Snijders P. J., Raaphorst P. M., Schrijnemakers H. F., Delius H., Gissmann L., Meijer C. J., Walboomers J. M. General primer polymerase chain reaction in combination with sequence analysis for identification of potentially novel human papillomavirus genotypes in cervical lesions. J Clin Microbiol. 1992 Jul;30(7):1716–1721. doi: 10.1128/jcm.30.7.1716-1721.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. zur Hausen H. Papillomaviruses in human cancer. Cancer. 1987 May 15;59(10):1692–1696. doi: 10.1002/1097-0142(19870515)59:10<1692::aid-cncr2820591003>3.0.co;2-f. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES