Skip to main content
PMC home page
Clinical Molecular Pathology logoLink to Clinical Molecular Pathology
. 1996 Aug;49(4):M236–M239. doi: 10.1136/mp.49.4.m236

Detection of human papillomavirus in vulval carcinoma using semi-nested PCR and restriction enzyme typing: a rapid and sensitive technique

N H Cartwright 1, L J Sant Cassia 1, A J Easton 1, A G Morris 1
PMCID: PMC408066  PMID: 16696082

Abstract

Aims—To develop a highly sensitive technique for the reliable detection and typing of human papillomavirus (HPV) DNA in clinical tissue.

Methods—A two step, semi-nested PCR was used with primers spanning the L1 region of the HPV genome and capable of detecting HPV DNA of all known HPV types. The clinical samples were typed by digestion of the 412 base pair PCR product with Rsa I, generating unique fragments for each HPV type. Thirteen samples were screened by this method, including nine vulval carcinoma samples and four wart samples from the penis and vulva.

Results—Experiments using DNA extracted from HPV DNA positive cell lines—that is, CaSki (HPV type 16) and HeLa (HPV type 18) established that the technique could detect as few as 50 HPV copies and that the predicted Rsa I fragments from HPV types 16 and 18 were generated. The predicted 412 base pair fragment was observed for all 13 clinical samples subjected to semi-nested PCR. Rsa I digestion of the product of the second round of PCR permitted the positive identification of the HPV type in most cases.

Conclusions—This technique provides an effective and rapid means of detecting HPV DNA, in most cases providing the HPV type. High risk HPV types were always detected in the nine vulval carcinoma samples analysed. The amount of tissue available from the biopsy specimens was small, confirming the sensitivity of the method.

Keywords: HPV

Keywords: semi-nested PCR

Keywords: vulval carcinoma

Full text

PDF
M236

Images in this article

M238Image
on p.M238
M238Image
on p.M238
M238Image
on p.M238

Selected References

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

  1. Arumainayagam J. T., Sumathipala A. H., Smallman L. A., Shahmanesh M. Flat condylomata of the penis presenting as patchy balanoposthitis. Genitourin Med. 1990 Aug;66(4):251–253. doi: 10.1136/sti.66.4.251. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bosch F. X., Manos M. M., Muñoz N., Sherman M., Jansen A. M., Peto J., Schiffman M. H., Moreno V., Kurman R., Shah K. V. Prevalence of human papillomavirus in cervical cancer: a worldwide perspective. International biological study on cervical cancer (IBSCC) Study Group. J Natl Cancer Inst. 1995 Jun 7;87(11):796–802. doi: 10.1093/jnci/87.11.796. [DOI] [PubMed] [Google Scholar]
  3. Chan K. W., Lam K. Y., Chan A. C., Lau P., Srivastava G. Prevalence of human papillomavirus types 16 and 18 in penile carcinoma: a study of 41 cases using PCR. J Clin Pathol. 1994 Sep;47(9):823–826. doi: 10.1136/jcp.47.9.823. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Chen S. L., Han C. P., Tsao Y. P., Lee J. W., Yin C. S. Identification and typing of human papillomavirus in cervical cancers in Taiwan. Cancer. 1993 Sep 15;72(6):1939–1945. doi: 10.1002/1097-0142(19930915)72:6<1939::aid-cncr2820720624>3.0.co;2-2. [DOI] [PubMed] [Google Scholar]
  5. Chow L. T., Nasseri M., Wolinsky S. M., Broker T. R. Human papillomavirus types 6 and 11 mRNAs from genital condylomata acuminata. J Virol. 1987 Aug;61(8):2581–2588. doi: 10.1128/jvi.61.8.2581-2588.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Crook T., Farthing A. Human papillomavirus and cervical cancer. 1993 Jan 20-Feb 2Br J Hosp Med. 49(2):131–132. [PubMed] [Google Scholar]
  7. Das B. C., Sharma J. K., Gopalkrishna V., Das D. K., Singh V., Gissmann L., zur Hausen H., Luthra U. K. A high frequency of human papillomavirus DNA sequences in cervical carcinomas of Indian women as revealed by Southern blot hybridization and polymerase chain reaction. J Med Virol. 1992 Apr;36(4):239–245. doi: 10.1002/jmv.1890360402. [DOI] [PubMed] [Google Scholar]
  8. 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]
  9. Franco E. L. Cancer causes revisited: human papillomavirus and cervical neoplasia. J Natl Cancer Inst. 1995 Jun 7;87(11):779–780. doi: 10.1093/jnci/87.11.779. [DOI] [PubMed] [Google Scholar]
  10. Friedrich E. G., Jr Vulvar vestibulitis syndrome. J Reprod Med. 1987 Feb;32(2):110–114. [PubMed] [Google Scholar]
  11. Haefner H. K., Tate J. E., McLachlin C. M., Crum C. P. Vulvar intraepithelial neoplasia: age, morphological phenotype, papillomavirus DNA, and coexisting invasive carcinoma. Hum Pathol. 1995 Feb;26(2):147–154. doi: 10.1016/0046-8177(95)90030-6. [DOI] [PubMed] [Google Scholar]
  12. Heinzel P. A., Chan S. Y., Ho L., O'Connor M., Balaram P., Campo M. S., Fujinaga K., Kiviat N., Kuypers J., Pfister H. Variation of human papillomavirus type 6 (HPV-6) and HPV-11 genomes sampled throughout the world. J Clin Microbiol. 1995 Jul;33(7):1746–1754. doi: 10.1128/jcm.33.7.1746-1754.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Hippeläinen M. I., Syrjänen S., Hippeläinen M. J., Saarikoski S., Syrjänen K. Diagnosis of genital human papillomavirus (HPV) lesions in the male: correlation of peniscopy, histology and in situ hybridisation. Genitourin Med. 1993 Oct;69(5):346–351. doi: 10.1136/sti.69.5.346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hippeläinen M., Yliskoski M., Saarikoski S., Syrjänen S., Syrjänen K. Genital human papillomavirus lesions of the male sexual partners: the diagnostic accuracy of peniscopy. Genitourin Med. 1991 Aug;67(4):291–296. doi: 10.1136/sti.67.4.291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hørding U. Human papillomavirus in epithelial neoplasia of the vulva and the uterine cervix. On the prevalence and possible significance of some genital HPV infections. Dan Med Bull. 1995 Apr;42(2):155–161. [PubMed] [Google Scholar]
  16. Hørding U., Kringsholm B., Andreasson B., Visfeldt J., Daugaard S., Bock J. E. Human papillomavirus in vulvar squamous-cell carcinoma and in normal vulvar tissues: a search for a possible impact of HPV on vulvar cancer prognosis. Int J Cancer. 1993 Sep 30;55(3):394–396. doi: 10.1002/ijc.2910550310. [DOI] [PubMed] [Google Scholar]
  17. Jacobs M. V., de Roda Husman A. M., van den Brule A. J., Snijders P. J., Meijer C. J., Walboomers J. M. Group-specific differentiation between high- and low-risk human papillomavirus genotypes by general primer-mediated PCR and two cocktails of oligonucleotide probes. J Clin Microbiol. 1995 Apr;33(4):901–905. doi: 10.1128/jcm.33.4.901-905.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kurman R. J., Toki T., Schiffman M. H. Basaloid and warty carcinomas of the vulva. Distinctive types of squamous cell carcinoma frequently associated with human papillomaviruses. Am J Surg Pathol. 1993 Feb;17(2):133–145. doi: 10.1097/00000478-199302000-00005. [DOI] [PubMed] [Google Scholar]
  19. Monk B. J., Burger R. A., Lin F., Parham G., Vasilev S. A., Wilczynski S. P. Prognostic significance of human papillomavirus DNA in vulvar carcinoma. Obstet Gynecol. 1995 May;85(5 Pt 1):709–715. doi: 10.1016/0029-7844(95)00045-s. [DOI] [PubMed] [Google Scholar]
  20. Okamoto M., Baba M., Kodama E., Sekine K., Takagi T., Kasukawa R., Shigeta S. Detection of hepatitis C virus genome in human serum by multi-targeted polymerase chain reaction. J Med Virol. 1993 Sep;41(1):6–10. doi: 10.1002/jmv.1890410103. [DOI] [PubMed] [Google Scholar]
  21. Planner R. S., Hobbs J. B. Intraepithelial and invasive neoplasia of the vulva in association with human papillomavirus infection. J Reprod Med. 1988 Jun;33(6):503–509. [PubMed] [Google Scholar]
  22. Prayson R. A., Stoler M. H., Hart W. R. Vulvar vestibulitis. A histopathologic study of 36 cases, including human papillomavirus in situ hybridization analysis. Am J Surg Pathol. 1995 Feb;19(2):154–160. doi: 10.1097/00000478-199502000-00004. [DOI] [PubMed] [Google Scholar]
  23. Reid R., Crum C. P., Herschman B. R., Fu Y. S., Braun L., Shah K. V., Agronow S. J., Stanhope C. R. Genital warts and cervical cancer. III. Subclinical papillomaviral infection and cervical neoplasia are linked by a spectrum of continuous morphologic and biologic change. Cancer. 1984 Feb 15;53(4):943–953. doi: 10.1002/1097-0142(19840215)53:4<943::aid-cncr2820530421>3.0.co;2-x. [DOI] [PubMed] [Google Scholar]
  24. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  25. Salam M. A., Rockett J., Morris A. General primer-mediated polymerase chain reaction for simultaneous detection and typing of human papillomavirus DNA in laryngeal squamous cell carcinomas. Clin Otolaryngol Allied Sci. 1995 Feb;20(1):84–88. doi: 10.1111/j.1365-2273.1995.tb00019.x. [DOI] [PubMed] [Google Scholar]
  26. Seah C. L., Chow V. T., Chan Y. C. Semi-nested PCR using NS3 primers for the detection and typing of dengue viruses in clinical serum specimens. Clin Diagn Virol. 1995 Aug;4(2):113–120. doi: 10.1016/0928-0197(94)00063-z. [DOI] [PubMed] [Google Scholar]
  27. Shamanin V., Delius H., de Villiers E. M. Development of a broad spectrum PCR assay for papillomaviruses and its application in screening lung cancer biopsies. J Gen Virol. 1994 May;75(Pt 5):1149–1156. doi: 10.1099/0022-1317-75-5-1149. [DOI] [PubMed] [Google Scholar]
  28. 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]
  29. Turner M. L., Marinoff S. C. Association of human papillomavirus with vulvodynia and the vulvar vestibulitis syndrome. J Reprod Med. 1988 Jun;33(6):533–537. [PubMed] [Google Scholar]
  30. Umpierre S. A., Kaufman R. H., Adam E., Woods K. V., Adler-Storthz K. Human papillomavirus DNA in tissue biopsy specimens of vulvar vestibulitis patients treated with interferon. Obstet Gynecol. 1991 Oct;78(4):693–695. [PubMed] [Google Scholar]
  31. Williamson A. L., Rybicki E. P. Detection of genital human papillomaviruses by polymerase chain reaction amplification with degenerate nested primers. J Med Virol. 1991 Mar;33(3):165–171. doi: 10.1002/jmv.1890330305. [DOI] [PubMed] [Google Scholar]
  32. Ylitalo N., Bergström T., Gyllensten U. Detection of genital human papillomavirus by single-tube nested PCR and type-specific oligonucleotide hybridization. J Clin Microbiol. 1995 Jul;33(7):1822–1828. doi: 10.1128/jcm.33.7.1822-1828.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. de Roda Husman A. M., Walboomers J. M., Meijer C. J., Risse E. K., Schipper M. E., Helmerhorst T. M., Bleker O. P., Delius H., van den Brule A. J., Snijders P. J. Analysis of cytomorphologically abnormal cervical scrapes for the presence of 27 mucosotropic human papillomavirus genotypes, using polymerase chain reaction. Int J Cancer. 1994 Mar 15;56(6):802–806. doi: 10.1002/ijc.2910560607. [DOI] [PubMed] [Google Scholar]

Articles from Clinical Molecular Pathology are provided here courtesy of BMJ Publishing Group

RESOURCES