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Journal of Clinical Microbiology, October 2001, p. 3530-3536, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3530-3536.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Nonisotopic Detection of Human Papillomavirus DNA in Clinical Specimens Using a Consensus PCR and a Generic Probe Mix in an Enzyme-Linked Immunosorbent Assay Format

J. R. Kornegay,1,* A. P. Shepard,1 C. Hankins,2,3 E. Franco,2 N. Lapointe,4,5 H. Richardson,2 The Canadian Women's HIV Study Group,dagger and F. Coutleé2,4,6

Roche Molecular Systems, Alameda, California,1 and Department of Epidemiology and Biostatistics, McGill University,2 Unité de Maladies Infectieuses, Direction de la Santé Publique de Montréal-Centre,3 Départements de Microbiologie et de Pédiatrie, Université de Montréal,4 Centre Maternel et Infantile sur le SIDA, Centre de Recherche de l'Hôpital Sainte-Justine, Hôpital Sainte-Justine,5 and Département de Microbiologie et Infectiologie, Hôpital Notre-Dame du Centre Hospitalier de l'Université de Montréal,6 Montreal, Quebec, Canada

Received 5 March 2001/Returned for modification 19 April 2001/Accepted 9 July 2001

We assessed the value of a new digoxigenin (DIG)-labeled generic probe mix in a PCR-enzyme-linked immunosorbent assay format to screen for the presence of human papillomavirus (HPV) DNA amplified from clinical specimens. After screening with this new generic assay is performed, HPV DNA-positive samples can be directly genotyped using a reverse blotting method with product from the same PCR amplification. DNA from 287 genital specimens was amplified via PCR using biotin-labeled consensus primers directed to the L1 gene. HPV amplicons were captured on a streptavidin-coated microwell plate (MWP) and detected with a DIG-labeled HPV generic probe mix consisting of nested L1 fragments from types 11, 16, 18, and 51. Coamplification and detection of human DNA with biotinylated beta -globin primers served as a control for both sample adequacy and PCR amplification. All specimens were genotyped using a reverse line blot assay (13). Results for the generic assay using MWPs and a DIG-labeled HPV generic probe mix (DIG-MWP generic probe assay) were compared with results from a previous analysis using dot blots with a radiolabeled nested generic probe mix and type-specific probes for genotyping. The DIG-MWP generic probe assay resulted in high intralaboratory concordance in genotyping results (88% versus 73% agreement using traditional methods). There were 207 HPV-positive results using the DIG-MWP method and 196 positives using the radiolabeled generic probe technique, suggesting slightly improved sensitivity. Only one sample failed to test positive with the DIG-MWP generic probe assay in spite of a positive genotyping result. Concordance between the two laboratories was nearly 87%. Approximately 6% of samples that were positive or borderline when tested with the DIG-MWP generic probe assay were not detected with the HPV type-specific panel, perhaps representing very rare or novel HPV types. This new method is easier to perform than traditional generic probe techniques and uses more objective interpretation criteria, making it useful in studies of HPV natural history.

* Corresponding author. Mailing address: Roche Molecular Systems, 1145 Atlantic Ave., Alameda, CA 94501. Phone: (510) 814-2749. Fax: (510) 522-1285. E-mail: janet.kornegay{at}roche.com.

dagger The Canadian Women's HIV Study Group includes the following investigators throughout Canada: principal investigators, Catherine Hankins and Normand Lapointe; Calgary, John Gill; Edmonton, Barbara Romanowski and Stephen Shafran; Halifax, Rob Grimshaw, David Haase, Lynn Johnston, and Wally Schlech; Hamilton, Stephan Landis, John Sellors, and Fiona Smaill; Montréal, François Beaudoin, Ngoc Biu, Alena Capek, Marc Boucher, Michel Chateauvert, Manon Coté, François Coutlée, Douglas Dalton, Gretty Deutsch, Julian Falutz, Diane Francoeur, Lisa Hallman, Eleanor Hew, Lina Karayan, Marina Klein, Louise Labrecque, Richard Lalonde, Christiane Lavoie, Catherine Lounsbury, John Macleod, Nicole Marceau, Gail Myhr, Grégoire Noel, Robert Piché, Manisha Raut, Chantal Rondeau, Jean-Pierre Routy, Karoon Samikian, Pierre Simard, Christina Smeja, Graham Smith, Paul-Pierre Tellier, and Emil Toma; Ottawa, Garry Garber and Garry Victor; Québec, Louise Côté, Edith Guilbert, Michel Morissette, Hélène Senay, and Sylvie Trottier; Toronto, Phil Berger, Lisa Friedland, Donna Keystone, Joan Murphy, Anne Phillips, Marion Powell, Anita Rachlis, Pat Rockman, Irving Salit, Cheryl Wagner, and Sharon Walmsey; Saskatoon, Kurt Williams; St. John, Ian Bowmer and Rory Windrim; Sudbury, Roger Sandre; Vancouver, Penny Ballem, David Burdge, Brian Conway, Mariane Harris, Deborah Money, Julio Montaner, Deborah Money, and Janice Veenhuizen.

Journal of Clinical Microbiology, October 2001, p. 3530-3536, Vol. 39, No. 10
0095-1137/01/$04.00+0   DOI: 10.1128/JCM.39.10.3530-3536.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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