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. 1995 Aug;33(8):1973–1978. doi: 10.1128/jcm.33.8.1973-1978.1995

Detection of human papillomavirus DNA in cervical lavage specimens by a nonisotopic consensus PCR assay.

F Coutlée 1, D Provencher 1, H Voyer 1
PMCID: PMC228319  PMID: 7559932

Abstract

A gene amplification method that combines PCR with an enzyme immunoassay (PCR-EIA) for quantitation of amplified DNA was developed for the detection of human papillomavirus (HPV). Samples were amplified with consensus primers MY09 and MY11. Amplified DNA products were reacted in solution with type-specific nested RNA probes labelled with digoxigenin-11-UTP. Hybrids were captured on a microtiter plate coated with an antidigoxigenin antibody. Bound DNA-RNA hybrids were quantitated by the addition of an alkaline phosphatase-labelled monoclonal antibody directed against DNA-RNA hybrids and a fluorogenic substrate. The detection limit of PCR-EIA was six copies of HPV type 18 DNA in the original specimen. The assay was used to assess HPV infection of the uterine cervixes of 65 women referred to a colposcopy clinic. In 66 cervicovaginal lavage specimens, all 23 HPV strains detected by a standard isotopic PCR assay were also detected by the PCR-EIA (sensitivity, 100%; 95% confidence interval, 85.2 to 100%). Forty-two of the 43 samples that did not contain HPV types 6/11, 16, 18, 31, 33, 35, and 45 were also negative by PCR-EIA, for a specificity of 97.7%. Low-level cross-reactivity was encountered between HPV types 18 and 45 as well as between types 33 and 58. PCR-EIA provides a convenient means of objectively measuring PCR-amplified HPV DNA from common genital HPV types.

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Selected References

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  1. 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]
  2. Bobo L., Coutlee F., Yolken R. H., Quinn T., Viscidi R. P. Diagnosis of Chlamydia trachomatis cervical infection by detection of amplified DNA with an enzyme immunoassay. J Clin Microbiol. 1990 Sep;28(9):1968–1973. doi: 10.1128/jcm.28.9.1968-1973.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Chan S. Y., Bernard H. U., Ong C. K., Chan S. P., Hofmann B., Delius H. Phylogenetic analysis of 48 papillomavirus types and 28 subtypes and variants: a showcase for the molecular evolution of DNA viruses. J Virol. 1992 Oct;66(10):5714–5725. doi: 10.1128/jvi.66.10.5714-5725.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Cherian T., Bobo L., Steinhoff M. C., Karron R. A., Yolken R. H. Use of PCR-enzyme immunoassay for identification of influenza A virus matrix RNA in clinical samples negative for cultivable virus. J Clin Microbiol. 1994 Mar;32(3):623–628. doi: 10.1128/jcm.32.3.623-628.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cole S. T., Danos O. Nucleotide sequence and comparative analysis of the human papillomavirus type 18 genome. Phylogeny of papillomaviruses and repeated structure of the E6 and E7 gene products. J Mol Biol. 1987 Feb 20;193(4):599–608. doi: 10.1016/0022-2836(87)90343-3. [DOI] [PubMed] [Google Scholar]
  6. Cole S. T., Streeck R. E. Genome organization and nucleotide sequence of human papillomavirus type 33, which is associated with cervical cancer. J Virol. 1986 Jun;58(3):991–995. doi: 10.1128/jvi.58.3.991-995.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Coutlee F., Viscidi R. P., Yolken R. H. Comparison of colorimetric, fluorescent, and enzymatic amplification substrate systems in an enzyme immunoassay for detection of DNA-RNA hybrids. J Clin Microbiol. 1989 May;27(5):1002–1007. doi: 10.1128/jcm.27.5.1002-1007.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Coutlée F., Bobo L., Abbass H., Dalabetta G., Hook N. E., Shah K., Viscidi R. P. Detection of HPV-16 in cell lines and cervical lavage specimens by a polymerase chain reaction-enzyme immunoassay assay. J Med Virol. 1992 May;37(1):22–29. doi: 10.1002/jmv.1890370105. [DOI] [PubMed] [Google Scholar]
  9. Coutlée F., Bobo L., Mayur K., Yolken R. H., Viscidi R. P. Immunodetection of DNA with biotinylated RNA probes: a study of reactivity of a monoclonal antibody to DNA-RNA hybrids. Anal Biochem. 1989 Aug 15;181(1):96–105. doi: 10.1016/0003-2697(89)90399-0. [DOI] [PubMed] [Google Scholar]
  10. Coutlée F., Saint-Antoine P., Olivier C., Voyer H., Kessous-Elbaz A., Berrada F., Bégin P., Giroux L., Viscidi R. Evaluation of infection with human immunodeficiency virus type 1 by using nonisotopic solution hybridization for detection of polymerase chain reaction-amplified proviral DNA. J Clin Microbiol. 1991 Nov;29(11):2461–2467. doi: 10.1128/jcm.29.11.2461-2467.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dartmann K., Schwarz E., Gissmann L., zur Hausen H. The nucleotide sequence and genome organization of human papilloma virus type 11. Virology. 1986 May;151(1):124–130. doi: 10.1016/0042-6822(86)90110-8. [DOI] [PubMed] [Google Scholar]
  12. Franco E. L. Viral etiology of cervical cancer: a critique of the evidence. Rev Infect Dis. 1991 Nov-Dec;13(6):1195–1206. doi: 10.1093/clinids/13.6.1195. [DOI] [PubMed] [Google Scholar]
  13. Goldsborough M. D., DiSilvestre D., Temple G. F., Lorincz A. T. Nucleotide sequence of human papillomavirus type 31: a cervical neoplasia-associated virus. Virology. 1989 Jul;171(1):306–311. doi: 10.1016/0042-6822(89)90545-x. [DOI] [PubMed] [Google Scholar]
  14. Guerrero E., Daniel R. W., Bosch F. X., Castellsagué X., Muñoz N., Gili M., Viladiu P., Navarro C., Zubiri M. L., Ascunce N. Comparison of ViraPap, Southern hybridization, and polymerase chain reaction methods for human papillomavirus identification in an epidemiological investigation of cervical cancer. J Clin Microbiol. 1992 Nov;30(11):2951–2959. doi: 10.1128/jcm.30.11.2951-2959.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. He Y., Coutlée F., Saint-Antoine P., Olivier C., Voyer H., Kessous-Elbaz A. Detection of polymerase chain reaction-amplified human immunodeficiency virus type 1 proviral DNA with a digoxigenin-labeled RNA probe and an enzyme-linked immunoassay. J Clin Microbiol. 1993 May;31(5):1040–1047. doi: 10.1128/jcm.31.5.1040-1047.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hildesheim A., Schiffman M. H., Gravitt P. E., Glass A. G., Greer C. E., Zhang T., Scott D. R., Rush B. B., Lawler P., Sherman M. E. Persistence of type-specific human papillomavirus infection among cytologically normal women. J Infect Dis. 1994 Feb;169(2):235–240. doi: 10.1093/infdis/169.2.235. [DOI] [PubMed] [Google Scholar]
  17. Höltke H. J., Kessler C. Non-radioactive labeling of RNA transcripts in vitro with the hapten digoxigenin (DIG); hybridization and ELISA-based detection. Nucleic Acids Res. 1990 Oct 11;18(19):5843–5851. doi: 10.1093/nar/18.19.5843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lorincz A. T., Reid R., Jenson A. B., Greenberg M. D., Lancaster W., Kurman R. J. Human papillomavirus infection of the cervix: relative risk associations of 15 common anogenital types. Obstet Gynecol. 1992 Mar;79(3):328–337. doi: 10.1097/00006250-199203000-00002. [DOI] [PubMed] [Google Scholar]
  19. Marich J. E., Pontsler A. V., Rice S. M., McGraw K. A., Dubensky T. W. The phylogenetic relationship and complete nucleotide sequence of human papillomavirus type 35. Virology. 1992 Feb;186(2):770–776. doi: 10.1016/0042-6822(92)90045-q. [DOI] [PubMed] [Google Scholar]
  20. Naghashfar Z. S., Rosenshein N. B., Lorincz A. T., Buscema J., Shah K. V. Characterization of human papillomavirus type 45, a new type 18-related virus of the genital tract. J Gen Virol. 1987 Dec;68(Pt 12):3073–3079. doi: 10.1099/0022-1317-68-12-3073. [DOI] [PubMed] [Google Scholar]
  21. 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]
  22. Schwarz E., Dürst M., Demankowski C., Lattermann O., Zech R., Wolfsperger E., Suhai S., zur Hausen H. DNA sequence and genome organization of genital human papillomavirus type 6b. EMBO J. 1983;2(12):2341–2348. doi: 10.1002/j.1460-2075.1983.tb01744.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Seedorf K., Krämmer G., Dürst M., Suhai S., Röwekamp W. G. Human papillomavirus type 16 DNA sequence. Virology. 1985 Aug;145(1):181–185. doi: 10.1016/0042-6822(85)90214-4. [DOI] [PubMed] [Google Scholar]
  24. Vermund S. H., Schiffman M. H., Goldberg G. L., Ritter D. B., Weltman A., Burk R. D. Molecular diagnosis of genital human papillomavirus infection: comparison of two methods used to collect exfoliated cervical cells. Am J Obstet Gynecol. 1989 Feb;160(2):304–308. doi: 10.1016/0002-9378(89)90430-4. [DOI] [PubMed] [Google Scholar]
  25. de Villiers E. M. Heterogeneity of the human papillomavirus group. J Virol. 1989 Nov;63(11):4898–4903. doi: 10.1128/jvi.63.11.4898-4903.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]

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