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Quantification of beta-human papillomavirus DNA by real-time PCR

Nature Protocols volume 5, pages 1–13 (2010)Cite this article

Abstract

Quantitative PCR with hybridization probes allows the reliable quantification of viral DNA sequences in clinical samples with a dynamic range and sensitivity that cannot be achieved with other methods. The technical background for the establishment of protocols is described and established protocols are presented to estimate the viral load per cell of frequently occurring betapapillomaviruses (HPV5, -8, -15, -20, -23, -24, -36 and -38) in skin tumors, healthy skin and hair bulbs. This approach accurately adjusts dilution series of reference DNA of different viral types relative to pUC18, which is crucial for comparative analyses and for interlaboratory standardization. The type-specific determination of beta-HPV DNA loads is an important research tool toward discrimination between low-level persistence and activated possibly pathologically relevant infections. The analysis of 24 samples, starting with DNA extraction and followed by HPV typing and quantification of—on average—three of the described HPV types takes about 2 d.

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Figure 1: Flowchart for quantification of beta-human papillomavirus DNA by real-time PCR.
Figure 2: Binding of primers and probes to viral DNA and amplimers.
Figure 3: Amplification plots of an HPV20 quantification experiment.

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References

  1. IARC. Human papillomaviruses. IARC Monogr. Eval. Carcinog. Risks Hum. 90, 245–259 (2007).

  2. Orth, G. Genetics of epidermodysplasia verruciformis: insights into host defense against papillomaviruses. Semin. Immunol. 18, 362–74 (2006).

    Article  CAS  Google Scholar 

  3. Berkhout, R.J. et al. Nested PCR approach for detection and typing of epidermodysplasia verruciformis-associated human papillomavirus types in cutaneous cancers from renal transplant recipients. J. Clin. Microbiol. 33, 690–695 (1995).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. Forslund, O., Antonsson, A., Nordin, P., Stenquist, B. & Hansson, B.G. A broad range of human papillomavirus types detected with a general PCR method suitable for analysis of cutaneous tumours and normal skin. J. Gen. Virol. 80 (Part 9): 2437–2443 (1999).

    Article  CAS  Google Scholar 

  5. Pfister, H. et al. High prevalence of epidermodysplasia verruciformis-associated human papillomavirus DNA in actinic keratoses of the immunocompetent population. Arch Dermatol. Res. 295, 273–279 (2003).

    Article  Google Scholar 

  6. Shamanin, V. et al. Human papillomavirus infections in nonmelanoma skin cancers from renal transplant recipients and nonimmunosuppressed patients. J. Natl. Cancer Inst. 88, 802–811 (1996).

    Article  CAS  Google Scholar 

  7. Boxman, I.L. et al. Detection of human papillomavirus DNA in plucked hairs from renal transplant recipients and healthy volunteers. J. Invest. Dermatol. 108, 712–715 (1997).

    Article  CAS  Google Scholar 

  8. Antonsson, A., Forslund, O., Ekberg, H., Sterner, G. & Hansson, B.G. The ubiquity and impressive genomic diversity of human skin papillomaviruses suggest a commensalic nature of these viruses. J. Virol. 74, 11636–11641 (2000).

    Article  CAS  Google Scholar 

  9. Astori, G. et al. Human papillomaviruses are commonly found in normal skin of immunocompetent hosts. J. Invest. Dermatol. 110, 752–755 (1998).

    Article  CAS  Google Scholar 

  10. Weissenborn, S.J., De Koning, M.N., Wieland, U., Quint, W.G. & Pfister, H.J. Intrafamilial transmission and family-specific spectra of cutaneous betapapillomaviruses. J. Virol. 83, 811–816 (2009).

    Article  CAS  Google Scholar 

  11. Antonsson, A., Karanfilovska, S., Lindqvist, P.G. & Hansson, B.G. General acquisition of human papillomavirus infections of skin occurs in early infancy. J. Clin. Microbiol. 41, 2509–2514 (2003).

    Article  Google Scholar 

  12. Hazard, K. et al. Cutaneous human papillomaviruses persist on healthy skin. J. Invest. Dermatol. 127, 116–119 (2007).

    Article  CAS  Google Scholar 

  13. de Koning, M.N. et al. Betapapillomaviruses frequently persist in the skin of healthy individuals. J. Gen. Virol. 88, 1489–1495 (2007).

    Article  CAS  Google Scholar 

  14. de Koning, M.N. et al. Prevalence and associated factors of betapapillomavirus infections in individuals without cutaneous squamous cell carcinoma. J. Gen. Virol. 90, 1611–1621 (2009).

    Article  CAS  Google Scholar 

  15. Harwood, C.A. et al. Increased risk of skin cancer associated with the presence of epidermodysplasia verruciformis human papillomavirus types in normal skin. Br. J. Dermatol. 150, 949–957 (2004).

    Article  CAS  Google Scholar 

  16. Struijk, L. et al. Presence of human papillomavirus DNA in plucked eyebrow hairs is associated with a history of cutaneous squamous cell carcinoma. J. Invest. Dermatol. 121, 1531–1535 (2003).

    Article  CAS  Google Scholar 

  17. Boxman, I.L. et al. Case–control study in a subtropical Australian population to assess the relation between non-melanoma skin cancer and epidermodysplasia verruciformis human papillomavirus DNA in plucked eyebrow hairs. The Nambour Skin Cancer Prevention Study Group. Int. J. Cancer 86, 118–121 (2000).

    Article  CAS  Google Scholar 

  18. Iftner, A. et al. The prevalence of human papillomavirus genotypes in nonmelanoma skin cancers of nonimmunosuppressed individuals identifies high-risk genital types as possible risk factors. Cancer Res. 63, 7515–7519 (2003).

    CAS  PubMed  Google Scholar 

  19. Asgari, M.M. et al. Detection of human papillomavirus DNA in cutaneous squamous cell carcinoma among immunocompetent individuals. J. Invest. Dermatol. 128, 1409–1417 (2008).

    Article  CAS  Google Scholar 

  20. Weissenborn, S.J. et al. Human papillomavirus-DNA loads in actinic keratoses exceed those in non-melanoma skin cancers. J. Invest. Dermatol. 125, 93–97 (2005).

    Article  CAS  Google Scholar 

  21. Dell'Oste, V. et al. High beta-HPV DNA loads and strong seroreactivity are present in epidermodysplasia verruciformis. J. Invest. Dermatol. 129, 1026–1034 (2009).

    Article  CAS  Google Scholar 

  22. Brink, A.A. et al. Development of a general-primer-PCR-reverse-line-blotting system for detection of beta and gamma cutaneous human papillomaviruses. J. Clin. Microbiol. 43, 5581–5587 (2005).

    Article  CAS  Google Scholar 

  23. de Koning, M. et al. Evaluation of a novel highly sensitive, broad-spectrum PCR-reverse hybridization assay for detection and identification of beta-papillomavirus DNA. J. Clin. Microbiol. 44, 1792–1800 (2006).

    Article  CAS  Google Scholar 

  24. Nindl, I. et al. Extension of the typing in a general-primer-PCR reverse-line-blotting system to detect all 25 cutaneous beta human papillomaviruses. J. Virol. Methods 146, 1–4 (2007).

    Article  CAS  Google Scholar 

  25. Gheit, T. et al. Development of a sensitive and specific multiplex PCR method combined with DNA microarray primer extension to detect betapapillomavirus types. J. Clin. Microbiol. 45, 2537–2544 (2007).

    Article  CAS  Google Scholar 

  26. Malnati, M.S. et al. A universal real-time PCR assay for the quantification of group-M HIV-1 proviral load. Nat. Protoc. 3, 1240–1248 (2008).

    Article  CAS  Google Scholar 

  27. Kubista, M. et al. The real-time polymerase chain reaction. Mol. Aspects Med. 27, 95–125 (2006).

    Article  CAS  Google Scholar 

  28. Kreuter, A. et al. Penile intraepithelial neoplasia is frequent in HIV-positive men with anal dysplasia. J. Invest. Dermatol. 128, 2316–2324 (2008).

    Article  CAS  Google Scholar 

  29. Vasiljevic, N. et al. Characterization of two novel cutaneous human papillomaviruses, HPV93 and HPV96. J. Gen. Virol. 88, 1479–1483 (2007).

    Article  CAS  Google Scholar 

  30. Vasiljevic, N., Hazard, K., Dillner, J. & Forslund, O. Four novel human betapapillomaviruses of species 2 preferentially found in actinic keratosis. J. Gen. Virol. 89, 2467–2474 (2008).

    Article  CAS  Google Scholar 

  31. Sutcliffe, J.G. Nucleotide sequence of the ampicillin resistance gene of Escherichia coli plasmid pBR322. Proc. Natl. Acad. Sci. USA 75, 3737–3741 (1978).

    Article  CAS  Google Scholar 

  32. Kwok, S. Procedures to minimize PCR-product carry-over. In PCR Protocols—A guide to Methods and Applications (eds. Innis, M.A., Gelfand, D.H., Sninsky, J.J. & White, T.J.) 142–145 (Academic Press, San Diego, California, 1990).

  33. Kwok, S. & Higuchi, R. Avoiding false positives with PCR. Nature 339, 237–238 (1989).

    Article  CAS  Google Scholar 

  34. Wittwer, T.C. et al. Continuous fluorescence monitoring of rapid cycle DNA amplification. BioTechniques 22, 130–138 (1997).

    Article  CAS  Google Scholar 

  35. Wittwer, T.C. et al. The LightCycler: a microvolume multisample fluorimeter with rapid temperature control. BioTechniques 22, 176–181 (1997).

    Article  CAS  Google Scholar 

  36. Mackay, J. & Landt, O. Real-time PCR fluorescent chemistries. In Protocols for Nucleic Acid Analysis by Nonradioactive Probes 2nd edn. Vol. 353 (eds. Hilario, E.& Mackay, J.) 237–261 (Humana Press, Clifton, New Jersey, 2007).

  37. Korbie, D.J. & Mattick, J.S. Touchdown PCR for increased specificity and sensitivity in PCR amplification. Nat. Protoc. 3, 1452–1456 (2008).

    Article  CAS  Google Scholar 

  38. Weissenborn, S.J. et al. Oncogenic human papillomavirus DNA loads in human immunodeficiency virus-positive women with high-grade cervical lesions are strongly elevated. J. Clin. Microbiol. 41, 2763–2767 (2003).

    Article  CAS  Google Scholar 

  39. Sambrook, J. & Russell, D.W. Molecular Cloning (Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2001).

  40. Kremsdorf, D., Jablonska, S., Favre, M. & Orth, G. Biochemical characterization of two types of human papillomaviruses associated with epidermodysplasia verruciformis. J. Virol. 43, 436–447 (1982).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. Pfister, H., Nürnberger, F., Gissmann, L. & zur Hausen, H. Characterization of a human papillomavirus from epidermodysplasia verruciformis lesions of a patient from Upper-volta. Int. J. Cancer 27, 645–650 (1981).

    Article  CAS  Google Scholar 

  42. Steger, G., Olszewsky, M., Stockfleth, E. & Pfister, H. Prevalence of antibodies to human papillomavirus type 8 in human sera. J. Virol. 64, 4399–4406 (1990).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. Kremsdorf, D. et al. Molecular cloning and characterization of the genomes of nine newly recognized human papillomavirus types associated with epidermodysplasia verruciformis. J. Virol. 52, 1013–1018 (1984).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. Kawashima, M., Favre, M., Jablonska, S., Obalek, S. & Orth, G. Characterization of a new type of human papillomavirus (HPV) related to HPV5 from a case of actinic keratosis. Virology 154, 389–394 (1986).

    Article  CAS  Google Scholar 

  45. Scheurlen, W., Gissmann, L., Gross, G. & zur Hausen, H. Molecular cloning of two new HPV types (HPV 37 and HPV 38) from a keratoacanthoma and a malignant melanoma. Int. J. Cancer 37, 505–510 (1986).

    Article  CAS  Google Scholar 

  46. Wieland, U. et al. Communication: papillomavirus DNA in basal cell carcinomas of immunocompetent patients: an accidental association? J. Invest. Dermatol. 115, 124–128 (2000).

    Article  CAS  Google Scholar 

  47. Saiki, R.K. et al. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science 230, 1350–1354 (1985).

    Article  CAS  Google Scholar 

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Acknowledgements

This study was supported by EC Grant QLK-CT-200201179. S.J.W. is supported by the 'Deutsche Krebshilfe,' EMBO and the Köln Fortune Program of the University of Cologne. U.W. was supported by the German Federal Ministry of Education and Research (BMBF) (Grant no. 01 KI 0771 (TP7)).

AUTHOR CONTRIBUTIONS

S.J.W. conceived and designed the protocol; S.J.W., M.J., U.W. and H.P. provided administrative, technical or material support; and S.J.W., U.W. and H.P. wrote the paper.

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Authors and Affiliations

  1. Institute of Virology, German National Reference Centre for Papilloma and Polyomaviruses, University of Cologne, Koeln, Germany

    Sönke J Weissenborn, Ulrike Wieland, Monika Junk & Herbert Pfister

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  1. Sönke J Weissenborn
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Correspondence to Herbert Pfister.

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Weissenborn, S., Wieland, U., Junk, M. et al. Quantification of beta-human papillomavirus DNA by real-time PCR. Nat Protoc 5, 1–13 (2010). https://doi.org/10.1038/nprot.2009.153

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