Direct sequencing of consensus primer generated PCR fragments of human papillomaviruses

doi:10.1016/0166-0934(93)90151-G

Journal of Virological Methods

Volume 43, Issue 3, August 1993, Pages 335-350

https://doi.org/10.1016/0166-0934(93)90151-G Get rights and content

Abstract

Consensus primers specific for the L1 and E1 regions were used to amplify HPV DNA fragments. These fragments were then directly sequenced using the consensus primers as the sequencing primers. The linking of PCR amplification, direct sequencing, and computer data bank analysis provides a new approach in the detection of different HPVs and has several advantages over existing methodology. These advantages include increased precision in the rapid characterization of known HPVs, detection of mutations, and identification of new HPV types.

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Cited by (30)

High prevalence of genital human papillomavirus type 52 and 58 infection in women attending gynecologic practitioners in South Taiwan
2006, Gynecologic Oncology
Citation Excerpt :
There are now several molecular techniques available for HPV genotyping. Direct sequencing and restriction fragment length polymorphism (RFLP) analyses are both common methods for HPV genotyping [10,11]. However, it is sometimes difficult to interpret the final results in cases with multiple infections.
We attempted to determine the prevalence of genital human papillomavirus (HPV) infection in women attending gynecologic practitioners in South Taiwan.
The population included 4383 women aged 16–78 seeking HPV testing at primary gynecologic practitioners regardless of their cervical cytology results. HPV DNA was identified from cervical swabs using semi-nested polymerase chain reaction with MY11, MY09/HMB01, and MY11/bioGP6+ primers. Genotyping for high-risk HPV (HR-HPV) was done separately by a HR-HPV chip, which contained 13 type-specific oligonucleotides on a nylon membrane.
The overall HPV prevalence was 19.3% (849/4383), 11.1% (488/4383) were confirmed as HR-HPV positive. Among the women with HR-HPV infection, HPV-16 was the most prevalent type (22.1%; 108/488), followed by HPV-52 (21.3%; 104/488), and HPV-58 (19.9%; 97/488). Multiple infections were detected in 73 women (15.0%; 73/488). For women with age 30 or younger, the overall HPV and HR-HPV prevalence were 32.0% and 20.7%, respectively, which were significantly higher than those of women age older than 30 (17.2% and 9.5%, P < 0.001). More multiple infections (22.1% vs. 12.4%) were also found in women with age 30 or younger (P = 0.021). However, the relative contribution of types to the overall HR-HPV positive among different age groups remains the same.
Our results showed an HPV prevalence that is similar compared with worldwide levels. HPV prevalence and multiple infections rate were decreasing across the age groups. Unlike most previous studies, the relative high prevalence of HPV 52 and 58 among South Taiwan women has important implications in vaccine prophylaxis.
Multiplex nested PCR (MNP) assay for the detection of 15 high risk genotypes of human papillomavirus
2005, Journal of Clinical Virology
Human papillomavirus (HPV) is now recognized as the causative agent in cervical cancer. The HPV genotypes that infect the genital region have been classified into high and low risk types according to their oncogenic potential. There is still uncertainty regarding rare HPV genotypes, however the types considered high risk in this study are: HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68 and 70.
We have set out to develop a multiplex nested PCR (MNP) assay with primers directed at the early region of the HPV genome to detect 15 high risk HPV (HRHPV) genotypes. Since it is known that the late region of HPV is lost on integration into the host cell genome, the primers are directed at the early region of the HPV genome so as to ensure the detection of integrated virus, in the absence of the episomal form of the virus.
Primers were designed to detect specifically the high risk HPV in the MNP assay. The MNP assay was compared to a generic mucosal HPV nested PCR and another nested HRHPV PCR assay. DNA sequencing was carried out on the samples tested and matched with the PCR results.
The MNP assay demonstrated that it was able to detect all 15 HRHPV types and was positive for more CIN1, CIN2 and CIN3 cases than the other nested HRHPV PCR. Further to this, the PCR product sizes differ for most of the HRHPV types detected in this system, so it is possible to type most of these HRHPV by the molecular size of the PCR products.
The MNP assay detects 15 currently recognized HRHPV and could be very useful, in conjunction with the Pap smear, as a screening assay or to help manage Pap smears of uncertain cytology.
A simple method for the detection and genotyping of high-risk human papillomavirus using seminested polymerase chain reaction and reverse hybridization
2005, Gynecologic Oncology
To develop a simple and cost-effective method for the detection and genotyping of high-risk human papillomaviruses (HPV) using seminested polymerase chain reaction (PCR) and reverse hybridization.
Cervical swabs for HPV testing were collected from 127 women with normal cervical cytology and 57 patients with cervical lesions of various degrees. After DNA isolation, PCR amplification was first carried out using MY11 and MY09/HMB01 primers, then labeled by seminested PCR using the first PCR products and MY11/bioGP6+ primers. One fifth of the second PCR products were resolved by gel electrophoresis. Genotyping for high-risk HPV was done separately, using the remaining products, by a high-risk HPV chip, which contained 13 type-specific oligonucleotides on a nylon membrane. The final result was detected by colorimetric change on the chip under direct visualization.
High-risk HPV DNA was detected in 19 (15%) of 127 women with normal cervical smear cytology, in 26 (89.7%) of 29 patients with cervical intraepithelial neoplasia (CIN), and in 27 (96.4%) of 28 patients with invasive cervical carcinoma. Multiple high-risk HPV infections were detected in five cases. HPV type 16 was the most frequent type of infection, comprising 34.5% and 53.6% of the patients with CIN and invasive carcinoma, respectively. The samples without a visible 190-bp band on electrophoresis exclusively showed negative hybridization results. This method could detect one to two copies of the HPV-16 genome derived from one SiHa cell. The overall sensitivity of HPV detection was 25 to 50 copies of HPV genome for each specimen. Thirteen high-risk types and twenty-four different types of HPV DNA showed specific hybridization without any cross-reaction.
Our results demonstrated the feasibility and optimistic prospects for this simple and cheap method of high-risk HPV genotyping. This technology can be easily set up in a routine molecular laboratory and would probably be of great value in cervical cancer prevention programs.
Nested PCR with the PGMY09/11 and GP5 <sup>+</sup>/6 <sup>+</sup> primer sets improves detection of HPV DNA in cervical samples
2004, Journal of Virological Methods
Based on epidemiological and research evidence, HPV has a causal role in cervical carcinogenesis. Several HPV detection methods exist to date; the most commonly used method for detection of genital HPVs consists of nested PCR using the MY09/11 and GP5⁺/6⁺ primer sets (MY/GP⁺). Recently, the PGMY09/11 primer set, a modified version of the MY09/11 primer set, was introduced for single PCR and was found to detect a wider range of HPV types. The next logical step was taken and the efficacy of nested PCR using the PGMY09/11 and GP5⁺/6⁺ primer sets (PGMY/GP⁺) to detect HPV in cervical samples was evaluated.
In this comparative study, nested PCR using the novel PGMY/GP⁺ primer set combination was found to be more type sensitive than the nested PCR with the MY/GP⁺ primer sets, detecting a wider range of HPV types, low copy HPVs, and better characterizing samples infected with multiple strains of HPV. Standardization and use of the PGMY/GP⁺ PCR system could aid physicians in providing more efficient HPV screening and better treatment for patients.
Enhancement of the innate and cellular immune response in patients with genital warts treated with topical imiquimod cream 5%
1999, Antiviral Research
The mechanism of action of imiquimod 5% cream applied topically to patients with genital warts was evaluated in a double-blind, placebo-controlled study. Imiquimod (16 patients) or placebo (three patients) was applied three times per week for up to 16 weeks. All imiquimod-treated patients had a ≥75% reduction in total wart area while only one of three placebo-treated patients had a similar reduction. Wart biopsies were taken at prestudy, week 6, and end of treatment. Polymerase chain reaction (PCR) for human papillomavirus (HPV) DNA and reverse transcriptase (RT)-PCR for messenger (m)RNAs were used to identify cytokines, cellular markers, viral gene products, and cell cycle markers in these biopsies. Treatment with imiquimod, an immune response modifier, stimulated significant increases in mRNA for interferon (IFN)-α, IFN-γ and 2′,5′ oligoadenylate synthetase (2′,5′-AS) as well as a tendency towards increases in tumor necrosis factor (TNF)-α and interleukin-12 p40. Significant increases in mRNA for CD4 and a trend toward increases in CD8 were also observed in imiquimod-treated patients, suggesting activation of a cell mediated immune response. Imiquimod administration was also associated with a significant decrease in viral load as measured by HPV DNA and L1 mRNA. The effects on HPV markers were accompanied by an apparent decrease in mRNA expression for markers of cell proliferation and an increase in mRNA for markers of keratinocyte differentiation and tumor suppressors.
Mastication of verruca vulgaris associated with esophageal papilloma: HPV-45 sequences detected in oral and cutaneous tissues
1997, Journal of the American Academy of Dermatology
Human papillomaviruses (HPVs) are double-stranded, circular, epitheliotropic DNA viruses of which nearly 70 types have been identified. Specific HPV types exhibit a predilection to infect certain sites; however, occurrence is not unique or restricted to these sites. HPV typing may also be helpful in determining the oncogenic potential of HPV lesions. The most common HPV types, 6 and 11, are associated with benign mucosal lesions, whereas types 18, 16, 31, and 33 are thought to confer a high rate of malignant transformation. We describe a patient with both palmar verrucae and esophageal papillomatosis that proved to be HPV type 45 by polymerase chain reaction. HPV 45 has a high homology to HPV 18 and is a member of the relatively new "high-risk" mucosal HPV family in terms of cervical oncogenic potential. To our knowledge, HPV 45 has never been reported in cutaneous warts or esophageal lesions.(J Am Acad Dermatol 1997;36:853-7.)

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Direct sequencing of consensus primer generated PCR fragments of human papillomaviruses

Abstract

Virology

Genital human papillomavirus infection in female university students as determined by a PCR-based method

J. Am. Med Assoc.

Hybridization methods other than PCR: an update

Amplification of human papillomavirus DNA sequences by using conserved primers

J. Clin. Microbiol.

Polymerase chain reaction-based methods for the detection of human papillomavirus DNA

Role of human papillomaviruses in human cancer

Cancer Res.

The use of polymerase reaction amplification for the detection of genital human papillomaviruses

Detection of human papillomavirus in cervical scrapes by the polymerase chain reaction in relation to cytology: possible implications for cervical cancer screening

Improved double stranded DNA sequencing using the linear polymerase chain reaction

Detection human papillomaviruses in cervicovaginal cells using polymerase chain reaction

Infect. Dis.