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Journal of Clinical Microbiology, February 2000, p. 688-695, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

A Novel and Rapid PCR-Based Method for Genotyping Human Papillomaviruses in Clinical Samples

Joshua H. Nelson,1 Gregory A. Hawkins,2,dagger Karin Edlund,3 Magnus Evander,3 Lennart Kjellberg,3 Göran Wadell,3 Joakim Dillner,4 Tsilya Gerasimova,5 Ann L. Coker,6 Lucia Pirisi,5 Daniel Petereit,7 and Paul F. Lambert1,*

McArdle Laboratory for Cancer Research1 and Department of Human Oncology,7 University of Wisconsin Medical School, and Epicentre Technologies,2 Madison, Wisconsin; Department of Virology, Umeåa,3 and Karolinska Institute, Stockholm,4 Sweden; and University of South Carolina School of Medicine5 and University of South Carolina School of Public Health,6 Columbus, South Carolina

Received 4 May 1999/Returned for modification 11 August 1999/Accepted 22 November 1999

Many human papillomavirus (HPV) genotypes are associated with cervical carcinoma. We demonstrate the utility of an innovative technique for genotyping of HPV in cervical tissue samples. This method provides an accurate means of identification of the specific HPV genotypes present in clinical specimens. By using the MY09-MY11 and the GP5+-GP6+ consensus primer pairs, HPV sequences were amplified by nested PCR from DNA isolated from cervical smear samples. This led to the production of an approximately 140-bp PCR product from the L1 (major capsid) gene of any of the HPVs present in the sample. PCR was performed with a deoxynucleoside triphosphate mixture which resulted in the incorporation of deoxyuridine into the amplified DNA product at positions where deoxythymidine would normally be incorporated at a frequency of about once or twice per strand. Following the PCR, the product was treated with an enzyme mix that contains uracil N-glycosylase (UNG) and endonuclease IV. UNG removes the uracil base from the nucleotide, and endonuclease IV cleaves the phosphodiester bond at this newly formed abasic site, producing fragments of various sizes. By having end labeled one of the amplification primers, a DNA ladder which is analogous to a "T-sequencing ladder" was produced upon electrophoresis of the products. By comparing this T-sequencing ladder to the known sequences of HPVs, the genotypes of unknown HPV isolates in samples were assigned. Data showing the utility of this technique for the rapid analysis of clinical samples are presented.

* Corresponding author. Mailing address: McArdle Laboratory for Cancer Research, 1400 University Ave., Madison, WI 53706. Phone: (608) 262-8533. Fax: (608) 262-2824. E-mail: lambert{at}oncology.wisc.edu.

dagger Present address: MWG Biotech Inc., High Point, NC 27265.

Journal of Clinical Microbiology, February 2000, p. 688-695, Vol. 38, No. 2
0095-1137/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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