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Journal of Clinical Microbiology, June 1999, p. 1852-1857, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

Nonisotopic Detection and Typing of Human Papillomavirus DNA in Genital Samples by the Line Blot Assay

François Coutlée,1,2,* Patti Gravitt,3,dagger Harriet Richardson,4 Catherine Hankins,4,5 Eduardo Franco,4 Normand Lapointe,1,6 Hélène Voyer,2 and The Canadian Women's HIV Study GroupDagger

Départements de Microbiologie-Immunologie et de Pédiatrie, Université de Montréal,1 Centre de Recherche et Département de Microbiologie et Infectiologie, Centre Hospitalier de l'Université de Montréal, Campus Notre-Dame,2 Unité de Maladies Infectieuses, Direction de la Santé Publique de Montréal-Centre,5 Department of Epidemiology and Biostatistics, McGill University,4 and Centre Maternel et Infantile sur le SIDA, Centre de Recherche de l'Hôpital Sainte-Justine, Hôpital Sainte-Justine,6 Montréal, Québec, Canada, and Roche Molecular Systems, Alameda, California3

Received 26 October 1998/Returned for modification 7 December 1998/Accepted 15 March 1999

The line blot assay, a gene amplification method that combines PCR with nonisotopic detection of amplified DNA, was evaluated for its ability to detect human papillomavirus (HPV) DNA in genital specimens. Processed samples were amplified with biotin-labeled primers for HPV detection (primers MY09, MY11, and HMB01) and for beta -globin detection (primers PC03 and PC04). Amplified DNA products were hybridized by a reverse blot method with oligonucleotide probe mixtures fixed on a strip that allowed the identification of 27 HPV genotypes. The line blot assay was compared to a standard consensus PCR test in which HPV amplicons were detected with radiolabeled probes in a dot blot assay. Two hundred fifty-five cervicovaginal lavage specimens and cervical scrapings were tested in parallel by both PCR tests. The line blot assay consistently detected 25 copies of HPV type 18 per run. The overall positivity for the DNA of HPV types detectable by both methods was 37.7% (96 of 255 samples) by the line blot assay, whereas it was 43.5% (111 of 255 samples) by the standard consensus PCR assay. The sensitivity and specificity of the line blot assay reached 84.7% (94 of 111 samples) and 98.6% (142 of 144 samples), respectively. The agreement for HPV typing between the two PCR assays reached 83.9% (214 of 255 samples). Of the 37 samples with discrepant results, 33 (89%) were resolved by avoiding coamplification of beta -globin and modifying the amplification parameters. With these modifications, the line blot assay compared favorably to an assay that used radiolabeled probes. Its convenience allows the faster analysis of samples for large-scale epidemiological studies. Also, the increased probe spectrum in this single hybridization assay permits more complete type discrimination.

* Corresponding author. Mailing address: Département de Microbiologie et Infectiologie, Centre Hospitalier de l'Université de Montréal, Campus Notre-Dame, 1560 Sherbrooke est, Montréal, Québec H2L 4M1, Canada. Phone: 514-281-6000, ext. 5162. Fax: 514-896-4607. E-mail: coutleef{at}sympatico.ca.

dagger Present address: Department of Epidemiology, Johns Hopkins University School of Hygiene and Public Health, Baltimore, MD 21205.

Dagger The Canadian Women's HIV study group includes the following investigators: Catherine Hankins, Normand Lapointe, John Gill, Barbara Romanowski, Stephen Shafran, Rob Grimshaw, David Haase, Wally Schlech, Stephen Landis, John Sellors, Fiona Smaill, Francois Beaudoin, Marc Boucher, Ngoc Bui, Michel Chateauvert, Manon Coté, François Coutlée, Douglas Dalton, Gretty Deutsch, Julian Falutz, Diane Francoeur, Lisa Hallman, Lina Karayan, Louise Labrecque, Richard Lalonde, Christiane Lavoie, Catherine Lounsbury, John Macleod, Nicole Marceau, Grégoire Noel, Grégoire Piché, Jean-Pierre Routy, Pierre Simard, Christina Smeja, Graham Smith, Pierre-Paul Tellier, Emil Toma, Garry Garber, Garry Victor, Louise Coté, Édith Guilbert, Michel Morissette, Hélène Senay, Sylvie Trottier, Phil Berger, Lisa Friedland, Donna Keystone, Joan Murphy, Anne Phillips, Marion Powell, Anita Rachlis, Pat Rockman, Irving Salit, Cheryl Wagner, Sharon Walmsey, Kurt Williams, Ian Bowmer, Rory Windrim, Roger Sandre, Penny Ballem, David Burdge, Brian Conway, Marianne Harris, Deborah Money, Julio Montaner, and Janice Veenhuizen.

Journal of Clinical Microbiology, June 1999, p. 1852-1857, Vol. 37, No. 6
0095-1137/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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