Summary
Polymerase chain reaction (PCR)-based assays that use consensus primers to detect DNA of a broad spectrum of human papillomavirus (HPV) types in a single assay belong to the most frequently used methods to detect HPV in clinical specimens. Here, we describe in detail one of these assays, the so-called GP5+/6+ PCR method, which can be used to detect and type HPV DNA in crude extracts of cervical scrapes and biopsy specimens. Following PCR with GP5+ and GP6+ primers, the latter of which is biotinylated at its 5′ end, the presence of DNA of any of the high-risk genotypes can easily be determined by an enzyme immunoassay (EIA). In this assay, PCR products are captured in streptavidin-coated wells of a microtiter plate, denatured by alkaline treatment, and hybridized to cocktails of digoxigenin-labeled oligonucleotides specific for high-risk or low-risk HPV types. The resulting hybrids can then be detected by alkaline phosphatase conjugated anti-digoxigenin polyclonal antibodies and substrate followed by optical density reading. Subsequently, EIA-positive PCR products can be typed by a reverse line blot genotyping procedure that, using a miniblotter device, enables typing of up to 39 samples with specific oligonucleotide probes for 37 different HPV (sub)types in a single assay.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bosch, F. X., Lorincz, A., Munoz, N., Meijer, C. J., and Shah, K. V. (2002) The causal relation between human papillomavirus and cervical cancer. J. Clin. Pathol. 55, 244–265.
Munoz, N., Bosch, F. X., de Sanjose, S., et al. (2003) Epidemiologic classification of human papillomavirus types associated with cervical cancer. N. Engl. J. Med. 348, 518–527.
Snijders, P. J., van den Brule, A. J, and Meijer, C. J. (2002) The clinical relevance of human papillomavirus testing: relationship between analytical and clinical sensitivity. J. Pathol. 201, 1–6.
Nobbenhuis, M. A., Walboomers, J. M., Helmerhorst, T. J., et al. (1999) Relation of human papillomavirus status to cervical lesions and consequences for cervicalcancer screening: a prospective study. Lancet 354, 20–25.
Nobbenhuis, M. A., Meijer, C. J., van den Brule, A. J., et al. (2001) Addition of high-risk HPV testing improves the current guidelines on follow-up after treatment for cervical intraepithelial neoplasia. Br. J. Cancer 84, 796–801.
Rozendaal, L., Walboomers, J. M., van der Linden, J. C., et al. (1996) PCR-based high-risk HPV test in cervical cancer screening gives objective risk assessment of women with cytomorphologically normal cervical smears. Int. J. Cancer 68, 766–769.
Rozendaal, L., Westerga, J., van der Linden, J. C., et al. (2000) PCR based high risk HPV testing is superior to neural network based screening for predicting incident CIN III in women with normal cytology and borderline changes. J. Clin. Pathol. 53, 606–611.
Jacobs, M. V., Snijders, P. J. F., Voorhorst, F. J., et al. (1999) Reliable high risk HPV DNA testing by polymerase chain reaction: an intermethod and intramethod comparison. J. Clin. Pathol. 52, 498–503.
Anh, P. T., Hieu, N. T., Herrero, R., et al. (2003) Human papillomavirus infection among women in South and North Vietnam. Int. J. Cancer 104, 213–220.
Sukvirach, S., Smith, J. S., Tunsakul, S., et al. (2003) Population-based human papillomavirus prevalence in Lampang and Songkla, Thailand. J. Infect. Dis. 187, 1246–1256
Jacobs, M. V., Snijders, P. J., van den Brule, A. J., Helmerhorst, T. J., Meijer, C. J., and Walboomers, J. M. (1997) A general primer GP5+/GP6(+)-mediated PCRenzyme immunoassay method for rapid detection of 14 high-risk and 6 low-risk human papillomavirus genotypes in cervical scrapings. J. Clin. Microbiol. 35, 791–795.
van den Brule, A. J., Pol, R., Fransen-Daalmeijer, N., Schouls, L. M., Meijer, C. J., and Snijders, P. J. (2002) 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.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2005 Humana Press Inc.
About this protocol
Cite this protocol
Snijders, P.J.F., van den Brule, A.J.C., Jacobs, M.V., Pol, R.P., Meijer, C.J.L.M. (2005). HPV DNA Detection and Typing in Cervical Scrapes. In: Davy, C., Doorbar, J. (eds) Human Papillomaviruses. Methods in Molecular Medicine, vol 119. Humana Press. https://doi.org/10.1385/1-59259-982-6:101
Download citation
DOI: https://doi.org/10.1385/1-59259-982-6:101
Publisher Name: Humana Press
Print ISBN: 978-1-58829-373-2
Online ISBN: 978-1-59259-982-0
eBook Packages: Springer Protocols