Improved Amplification of Genital Human Papillomaviruses

P Gravitt

C Peyton

T Alessi

C Wheeler

Department of Human Genetics, Roche Molecular Systems, Alameda, California^{; Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, New Mexico}^{; Départements de Microbiologie-Infectiologíe, CHUM, Montréal, Québec, Canada}^{; Environmental Epidemiology Branch, National Cancer Institute, Rockville, Maryland}^{; and Department of Pathology, Kaiser Permanente, Portland, Oregon}⁵

^{Corresponding author. Present address: 2810 St. Paul St., #1, Baltimore, MD 21218. Phone: (410) 889-5456. Fax: (301) 402-0916. E-mail:}ude.hpshj@ttivargp.

Received 1999 Jul 26; Revisions requested 1999 Sep 8; Accepted 1999 Oct 5.

Abstract

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 χ^{= 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.

Abstract

L1 consensus primer PCR systems, particularly the MY09-MY11 (MY09/11) and GP5+-GP6+ (GP5+/6+) primer systems (1, 4, 9, 13), have been widely used to study the natural history of human papillomaviruses (HPVs) and their role in the development of genital cancer, particularly of the uterine cervix (8, 10, 18). The MY09/11 HPV DNA detection system was used to show convincingly for the first time that the determinants of infection with HPV were the same as those for cervical cancer, namely, the sexual behavior variables such as increased number of lifetime sexual partners (11). Furthermore, both consensus primer methods have been used in a number of important studies that show unequivocally the associated risk of infection with certain types of HPV with the development of cervical cancer (12, 15). The sensitivities of these methods and their ability to amplify and detect greater than 25 of the HPV genotypes known to infect the genital mucosa have provided researchers with an extremely valuable tool which has been considered a “gold standard” for HPV detection for the last several years. However, despite the progress toward the understanding of HPV-associated disease facilitated by the use of these consensus primer systems, limitations are still evident, particularly in regard to the variability of detection sensitivity among specific HPV types (17).

At the time that the MY09/11 primer system was designed, only 5 of the 20 or more known genital HPV genotype sequences had been reported; specifically, HPV types 6, 11, 16, 18, and 33 (13). The primers were thus designed in a conserved region of the L1 open reading frame with the intent of amplifying in a single reaction both the five genotypes whose sequences are known and, presumptively, other genital HPVs with shared sequence homology in this region. The chosen regions were not entirely homologous even among the five original HPV types, and positions with nucleotide base heterogeneity were accommodated by inclusion of degenerate base sites. The resultant degenerate primers comprised a mixture of 24 unique oligonucleotide sequences. Over the next decade studies with these primers for amplification and detection of HPV from genital samples demonstrated the ability of the primers to amplify a spectrum of more than 30 genital HPV types, albeit with various levels of sensitivity (2). Only a single modification to the original primer set was made, wherein an extra, sequence-specific oligonucleotide (HMB01) directed to the minus strand of HPV type 51 (HPV-51) was included to facilitate the amplification of this important, cancer-associated type of HPV (7). The MY09/11 system referred to in this paper is inclusive of the HMB01 primer.

The nature of the synthesis of a mixture of oligonucleotides with degenerate base sequences relies on the presumed random addition of one of two or more nucleotide bases at the position of degeneracy. The random insertion of bases at degenerate positions is not a controlled process, such that an equal proportion of each sequence combination cannot be guaranteed. Furthermore, no analytical method for the verification of sequence proportions was readily available for quality control purposes, so that functional testing with each HPV type as a template was required to ensure the comparability of different lots of primers. Results from our own laboratories (P. E. Gravitt and F. Coutlée, unpublished data) indicate differences in type-specific amplification efficiencies among separate syntheses of the MY09/11 degenerate primers (data not shown).

We sought to improve the reproducibility and sensitivity of the MY09/11 HPV amplification system by developing a set of oligonucleotide pools, PGMY09 and PGMY11, based on the same primer binding regions used for MY09/11. Rather than using the degenerate primer method, we grouped virus types together by sequence homology in each of the two primer binding regions. From these groupings, we designed a set of 5 upstream oligonucleotides comprising the PGMY11 primer pool and a set of 13 downstream oligonucleotides comprising the PGMY09 primer pool (PGMY09/11 primer system). These primers were used in amplification reactions similar to the standard MY09/11 PCR protocols, and we continued to coamplify HPV with the internal β-globin control using the primer pair PC04 and GH20. We compared the performance of this new set of L1 consensus primers to that of the standard MY09/11 system for the amplification and detection of HPV from cervical cell samples.

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