Improved Amplification of Genital Human Papillomaviruses
Open accessAbstract
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 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.
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The PGMY primers used in PG are degenerate to identify potentially unknown genotypes.
“The PGMY primers used in PG are degenerate to identify potentially unknown genotypes (13).”
From Comparison of Seegene Anyplex II HPV28 with the PGMY-CHUV Assay for Human Papillomavirus Genotyping (2014) · cites this paper as [13]
The Abstract says PGMY09/11 accommodates sequence variation through pools of distinct primers, explicitly avoiding degenerate bases. The Discussion likewise describes eliminating the need for degenerate primer synthesis. The paper describes the earlier MY09/11 primers, not PGMY, as degenerate. (LLM, conf=0.90)
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Experimental — accuracy not yet measuredMany published HPV genotyping assays rely on endpoint multiplex PCR followed by reverse hybridization against a panel of type-specific probes immobilized on membranes or beads.
“Many published HPV genotyping assays rely on endpoint multiplex PCR followed by reverse hybridization against a panel of type-specific probes immobilized on membranes or beads (12)(13)(14)(15)(16)(17)(18)(19).”
From Comparison of Seegene Anyplex II HPV28 with the PGMY-CHUV Assay for Human Papillomavirus Genotyping (2014) · cites this paper as [13]
The Materials and Methods section describes PCR with pooled primers followed by hybridization of the products to an immobilized HPV probe array for genotyping. It does not identify the array as membrane- or bead-based, or establish that many published assays use this workflow. (LLM, conf=0.84)
Experimental — accuracy not yet measured
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- Authors
- Gravitt, P. E., Peyton, C. L., Alessi, T. Q., Wheeler, C. M., Coutlée, F., Hildesheim, A., Schiffman, M. H., Scott, D. R., & Apple, R. J.
- Affiliations
- <!--label omitted: 1-->Department of Human Genetics, Roche Molecular Systems, Alameda, California1;
- University of New Mexico (US)
- <!--label omitted: 3-->Départements de Microbiologie-Infectiologı́e, CHUM, Montréal, Québec, Canada3;
- National Cancer Institute (US)
- Kaiser Permanente (US)
- Venue
- Journal of Clinical Microbiology
- Publisher
- American Society for Microbiology
- Published
- 2000-01
- Volume
- 38
- Issue
- 1
- Pages
- 357-361
- ISSN
- 0095-1137 (ISSN-L)
- 1098-660X
- PMC
- PMC88724
- OpenAlex
- W2096478290
- Cited by
- 1,137
- References
- 1
- Citations by year
- 2026: 11 · 2025: 20 · 2024: 16 · 2023: 21 · 2022: 16 · 2021: 26 · 2020: 27 · 2019: 29 · 2018: 33 · 2017: 33 · 2016: 45 · 2015: 58 · 2014: 69 · 2013: 75 · 2012: 69
- FWCI
- 19.87
- Type
- journal article
- Language
- en
- Open access
- green
- europepmc.org
- Locations
- Journal of Clinical Microbiology (publishedVersion)
- Europe PMC (PubMed Central) (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Health Sciences › Medicine › Epidemiology › Cervical Cancer and HPV Research
- Life Sciences › Immunology and Microbiology › Microbiology › Reproductive tract infections research
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Keywords
- genital human papillomavirus, HPV genotyping, primer mismatch, L1 open reading frame, multiple HPV infection, HPV prevalence, cervicovaginal lavage
- MeSH terms
- Base Sequence, Cervix Uteri, Female, Genital Diseases, Female, Humans, Therapeutic Irrigation, Male, Tumor Virus Infections, Vagina, Polymerase Chain Reaction, Consensus Sequence, DNA Primers, Papillomaviridae, Papillomavirus Infections
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