Detection and Typing of Human Papillomaviruses Combining Different Methods: Polymerase Chain Reaction, Restriction Fragment Length Polymorphism, Line Probe Assay and Sequencing


The identification of the etiological factor of many cervical precancerous lesions and cervical cancer, the human papillomavirus (HPV) is widely used. In this study, we evaluated the consensus and type-specific (TS) polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), line probe assay (LiPA, Innogenetics) and sequencing to determine the HPV types in cervical specimens. Out of 690 High-grade Squamous Intraepithelial Lesion (HSIL) samples, 86.7% were HPV positive and 13.3% HPV negative by consensus primers (MY09/MY11, L1C1/L1C2-1/L1C2-2 and/or GP5/6) directed PCR. Out of 598 HPV positive samples, 85.3% were typed by TS-PCR being HPV 6/11, 16, 18, 31 and/or 33, while 14.7% remained untyped. The most prevalent HPV type in the study group was HPV 16, identified in 35.5% cases, while HPV 31 was the second most frequent HPV type with a prevalence of 10.5%. They were followed by HPV types 6/11, 33 and 18 with a prevalence of 7.4%, 6.2% and 4.9%, respectively. Multiple HPV infections with two or more HPV types (6/11, 16, 18, 31 and/or 33) were found in 9.4% cases. A subset of 88 samples was further typed by RFLP and LiPA to determine the rare HPV types in HSIL samples. The most frequent low abundant HPV types in single infections in decreasing order were HPV 53, 58, 66, 56 and 52, while HPV 51 was the most frequent low abundant HPV type found in multiple HPV infections. Multiple HPV infections were mostly found by LiPA in 27.3% cases versus 14.8% cases found by RFLP. The perfect agreement between RFLP and LiPA assay pair was observed only for HPV types 16, 18, 34 and 59 (kappa value of 1). For other HPV types, the inter-assay agreement ranged from very good to no agreement indicating that neither assay is perfect. Sequencing was performed for 33 samples in cases where both RFLP and LiPA were inconclusive. Sequencing was shown to be a very good method in case of single HPV infection but not applicable in case of multiple HPV infections. Both RFLP and LiPA are good assays for epidemiological studies, although RFLP being cumbersome and time-consuming is less applicable than LiPA. Careful consideration has to be made before the implementation of either HPV typing methods in clinical laboratories.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3



high-grade squamous intraepithelial lesion




human papillomavirus


line probe assay (Innogenetics)


polymerase chain reaction


restriction fragment length polymorphism




  1. 1.

    zur Hausen H (2002) Papillomaviruses and cancer: from basic studies to clinical application. Nat Rev Cancer 2:342–350

    PubMed  Article  Google Scholar 

  2. 2.

    Muñoz N, Castellsagué X, de González AB et al (2006) Chapter 1: HPV in the etiology of human cancer. Vaccine 24:S1–S10

    Article  Google Scholar 

  3. 3.

    Gillison ML, Lowy DR (2004) A causal role for human papillomavirus in head and neck cancer. Lancet 363:1488–1489

    PubMed  Article  CAS  Google Scholar 

  4. 4.

    de Villiers EM, Fauquet C, Broker TR et al (2004) Classification of papillomaviruses. Virology 324:17–27

    PubMed  Article  Google Scholar 

  5. 5.

    Walboomers JM, Jacobs MV, Manos MM et al (1999) Human papillomavirus is a necessary cause of invasive cervical cancer worldwide. J Pathol 189:12–19

    PubMed  Article  CAS  Google Scholar 

  6. 6.

    Muñoz N, Bosch FX, de Sanjosé S et al (2003) Epidemiologic classification of human papillomavirus types associated with cervical cancer. N Engl J Med 348:518–527

    PubMed  Article  Google Scholar 

  7. 7.

    Muñoz N, Bosch FX, Castellsagué X et al (2004) Against which human papillomavirus types shall we vaccinate and screen? The international perspective. Int J Cancer 111:278–285

    PubMed  Article  Google Scholar 

  8. 8.

    Grce M, Husnjak K, Bozikov J et al (2001) Evaluation of genital human papillomavirus infections by polymerase chain reaction among Croatian women. Anticancer Res 21:579–584

    PubMed  CAS  Google Scholar 

  9. 9.

    Bosch FX, Lorincz A, Muñoz N et al (2002) The causal relation between human papillomavirus and cervical cancer. J Clin Pathol 55:244–265

    PubMed  CAS  Google Scholar 

  10. 10.

    Iftner T, Villa LL (2003) Chapter 12: Human papillomavirus technologies. J Natl Cancer Inst Monogr 31:80–88

    PubMed  Google Scholar 

  11. 11.

    van den Brule AJ, Meijer CJ, Bakels V et al (1990) Rapid detection of human papillomavirus in cervical scrapes by combined general primer-mediated and type-specific polymerase chain reaction. J Clin Microbiol 28:2739–2743

    PubMed  Google Scholar 

  12. 12.

    Soler C, Allibert P, Chardonnet Y et al (1991) Detection of human papillomavirus types 6, 11, 16 and 18 in mucosal and cutaneous lesions by the multiplex polymerase chain reaction. J Virol Methods 35:143–157

    PubMed  Article  CAS  Google Scholar 

  13. 13.

    Gravitt PE, Peyton CL, Apple RJ et al (1998) Genotyping of 27 human papillomavirus types by using L1 consensus PCR products by a single-hybridization, reverse line blot detection method. J Clin Microbiol 36:3020–3027

    PubMed  CAS  Google Scholar 

  14. 14.

    Kleter B, van Doorn LJ, Schrauwen L et al (1999) Development and clinical evaluation of a highly sensitive PCR-reverse hybridization line probe assay for detection and identification of anogenital human papillomavirus. J Clin Microbiol 37:2508–2517

    PubMed  CAS  Google Scholar 

  15. 15.

    van den Brule AJ, Pol R, Fransen-Daalmeijer N et al (2000) GP5+/6+PCR followed by reverse line blot analysis enables rapid and high-throughput identification of human papillomavirus genotypes. J Clin Microbiol 40:779–787

    Article  Google Scholar 

  16. 16.

    Ovanin-Rakić A, Pajtler M, Stanković T et al (2003) [Classification of cervical cytological smears “Zagreb 2002”.Modification of the classification “Zagreb 1990” and “NCI Bethesda 2001”]. Gynaecol Perinatol 12:148–153 (In Croatian)

    Google Scholar 

  17. 17.

    Milutin-Gašperov N, Sabol I, Halec G et al (2007) Retrospective study of the prevalence of high-risk human papillomaviruses among Croatian women. Coll Antropol Suppl 2:89–96

    Google Scholar 

  18. 18.

    Maniatis T, Frisch EF, Sambrook J (eds) (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York

  19. 19.

    Grce M, Husnjak K, Magdić L et al (1997) Detection and typing of human papillomaviruses by polymerase chain reaction in cervical scrapes of Croatian women with abnormal cytology. Eur J Epidemiol 13:645–651

    PubMed  Article  CAS  Google Scholar 

  20. 20.

    Husnjak K, Grce M, Magdić L et al (2000) Comparison of five different polymerase chain reaction methods for detection of human papillomavirus in cervical cell specimens. J Virol Methods 88:125–134

    PubMed  Article  CAS  Google Scholar 

  21. 21.

    Bell DA, Taylor JA, Paulson DF et al (1993) Genetic risk and carcinogen exposure: a common inherited defect of the carcinogen-metabolism gene glutathione S-transferase M1 (GSTM1) that increases susceptibility to bladder cancer. J Natl Cancer Inst 85:1159–1164

    PubMed  Article  CAS  Google Scholar 

  22. 22.

    Ting Y, Manos MM (1990) Detection and typing of genital human papillomaviruses. In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds) PCR protocols: a guide to methods and applications. Academic, San Diego, p 356

    Google Scholar 

  23. 23.

    Meyer T, Arndt R, Stockfleth E et al (1995) Strategy for typing human papillomaviruses by RFLP analysis of PCR products and subsequent hybridization with a generic probe. Biotechniques 19:632–639

    PubMed  CAS  Google Scholar 

  24. 24.

    Bernard HU, Chan SY, Manos MM et al (1994) Identification and assessment of known and novel human papillomaviruses by polymerase chain reaction amplification, restriction fragment length polymorphisms, nucleotide sequence, and phylogenetic algorithms. J Infect Dis 170:1077–1085

    PubMed  CAS  Google Scholar 

  25. 25.

    Quint WG, Scholte G, van Doorn LJ et al (2001) Comparative analysis of human papillomavirus infections in cervical scrapes and biopsy specimens by general SPF(10) PCR and HPV genotyping. J Pathol 194:51–58

    PubMed  Article  CAS  Google Scholar 

  26. 26.

    Altschul SF, Gish W, Miller W et al (1990) Basic local alignment search tool. J Mol Biol 215:403–410

    PubMed  CAS  Google Scholar 

  27. 27.

    Clifford GM, Smith JS, Aguado T et al (2003) Comparison of HPV type distribution in high-grade cervical lesions and cervical cancer: a meta-analysis. Br J Cancer 89:101–105

    PubMed  Article  CAS  Google Scholar 

  28. 28.

    Smith JS, Lindsay L, Hoots B et al (2007) Human papillomavirus type distribution in invasive cervical cancer and high-grade cervical lesions: a meta-analysis update. Int J Cancer 121:621–632

    PubMed  Article  CAS  Google Scholar 

  29. 29.

    Perrons C, Kleter B, Jelley R et al (2002) Detection and genotyping of human papillomavirus DNA by SPF10 and MY09/11 primers in cervical cells taken from women attending a colposcopy clinic. J Med Virol 67:246–252

    PubMed  Article  CAS  Google Scholar 

  30. 30.

    van Doorn LJ, Quint W, Kleter B et al (2002) Genotyping of human papillomavirus in liquid cytology cervical specimens by the PGMY line blot assay and the SPF(10) line probe assay. J Clin Microbiol 40:979–983

    PubMed  Article  Google Scholar 

  31. 31.

    Gillio-Tos A, De Marco L, Ghisetti V et al (2006) Human papillomavirus typing with GP5+/6+polymerase chain reaction reverse line blotting and with commercial type-specific PCR kits. J Clin Virol 36:126–132

    PubMed  Article  CAS  Google Scholar 

  32. 32.

    Sabol I, Salakova M, Smahelova J et al (2008) Evaluation of different techniques for the identification of human papillomavirus types of low prevalence. J Clin Microbiol 46:1606–1613 doi:10.1128/JCM.02328-07

    PubMed  Article  Google Scholar 

Download references


This work was supported by grant numbers 098-0982464-2510 and 101-0982464-2277 from the Ministry of Science, Technology and Sport of the Republic of Croatia. The authors thank Jasminka Golubić Talić for expert technical assistance.

Author information



Corresponding author

Correspondence to Magdalena Grce.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Milutin Gašperov, N., Sabol, I., Matovina, M. et al. Detection and Typing of Human Papillomaviruses Combining Different Methods: Polymerase Chain Reaction, Restriction Fragment Length Polymorphism, Line Probe Assay and Sequencing. Pathol. Oncol. Res. 14, 355–363 (2008).

Download citation


  • Human papillomavirus
  • Consensus polymerase chain reaction
  • Restriction fragment length polymorphism
  • Line probe assay
  • Sequencing
  • Type-specific polymerase chain reaction