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Journal of Clinical Microbiology, January 2000, p. 357-361, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Improved Amplification of Genital Human Papillomaviruses

P. E. Gravitt,1,* C. L. Peyton,2 T. Q. Alessi,2 C. M. Wheeler,2 F. Coutlée,3 A. Hildesheim,4 M. H. Schiffman,4 D. R. Scott,5 and R. J. Apple1

Department of Human Genetics, Roche Molecular Systems, Alameda, California1; Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, New Mexico2; Départements de Microbiologie-Infectiologíe, CHUM, Montréal, Québec, Canada3; Environmental Epidemiology Branch, National Cancer Institute, Rockville, Maryland4; and Department of Pathology, Kaiser Permanente, Portland, Oregon5

Received 26 July 1999/Returned for modification 8 September 1999/Accepted 5 October 1999

Genital human papillomaviruses (HPVs) are commonly detected from clinical samples by consensus PCR methods. Two commonly used primer systems, the MY09-MY11 (MY09/11) primers and the GP5+-GP6+ (GP5+/6+) primers, amplify a broad spectrum of HPV genotypes, but with various levels of sensitivity among the HPV types. Analysis of the primer-target sequence homology for the MY09/11 primers showed an association between inefficient amplification of HPV types and the number and position of mismatches, despite accommodation of sequence variation by inclusion of degenerate base sites. The MY09/11 primers were redesigned to increase the sensitivity of amplification across the type spectrum by using the same primer binding regions in the L1 open reading frame. Sequence heterogeneity was accommodated by designing multiple primer sequences that were combined into an upstream pool of 5 oligonucleotides (PGMY11) and a downstream pool of 13 oligonucleotides (PGMY09), thereby avoiding use of degenerate bases that yield irreproducible primer syntheses. The performance of the PGMY09-PGMY11 (PGMY09/11) primer system relative to that of the standard MY09/11 system was evaluated with a set of 262 cervicovaginal lavage specimens. There was a 91.5% overall agreement between the two systems (kappa = 0.83; P < 0.001). The PGMY09/11 system appeared to be significantly more sensitive than the MY09/11 system, detecting an additional 20 HPV-positive specimens, for a prevalence of 62.8% versus a prevalence of 55.1% with the MY09/11 system (McNemar's chi 2 = 17.2; P < 0.001). The proportion of multiple infections detected increased with the PGMY09/11 system (40.0 versus 33.8% of positive infections). HPV types 26, 35, 42, 45, 52, 54, 55, 59, 66, 73, and MM7 were detected at least 25% more often with the PGMY09/11 system. The PGMY09/11 primer system affords an increase in type-specific amplification sensitivity over that of the standard MY09/11 primer system. This new primer system will be useful in assessing the natural history of HPV infections, particularly when the analysis requires HPV typing.

* Corresponding author. Present address: 2810 St. Paul St., #1, Baltimore, MD 21218. Phone: (410) 889-5456. Fax: (301) 402-0916. E-mail: pgravitt{at}jhsph.edu.

Journal of Clinical Microbiology, January 2000, p. 357-361, Vol. 38, No. 1
0095-1137/0/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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