Detection of Human Papillomaviruses in Cervical Neoplasias Using Multiple Sets of Generic Polymerase Chain Reaction Primers

Kado, Satoshi; Kawamata, Yasutaka; Shino, Yuji; Kasai, Tokuzo; Kubota, Kouichi; Iwasaki, Hiromichi; Fukazawa, I.; Takano, Hajime; Nunoyama, T; Mitsuhashi, Akira; Sekiya, Souei; Shirasawa, Hiroshi

doi:10.1006/gyno.2000.6116

Cited by 33 publications

(25 citation statements)

References 16 publications

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“…As expression of the E6 and E7 genes is directly associated with carcinogenesis, detection of these targets may provide more relevant biological and pathological information. E6 or E7 genes have been selected as amplification targets for HPV typing (16,18), and studies have shown that in cervical cancer samples that were positive for E6, the L1 gene could not be detected in about 30% of the cases (8,9). One potential disadvantage of targeting the E6/E7 region is the number of base pair variants in this region that could impact detection.…”

Section: Discussionmentioning

confidence: 99%

Simultaneous Amplification and Identification of 25 Human Papillomavirus Types with Templex Technology

Han

Swan

Smith³

et al. 2006

J Clin Microbiol

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The majority of existing human papillomavirus (HPV) genotyping assays are based on multiplex PCR using consensus or degenerate primers. We developed a Templex HPV assay that simultaneously detects and identifies 25 common HPV genotypes in a single-tube reaction using type-specific primers for the HPV-specific E6 and E7 genes. The analytical sensitivities of the Templex assay for HPV type 16 (HPV-16), -18, and -56 were 20, 100, and 20 copies per reaction mixture, respectively. The Templex assay provides semiquantitative information on each type when multiple HPV types coexist in one reaction. We tested 109 clinical cervical specimens previously evaluated with the Digene HC2 high-risk HPV DNA test and found 95.4% concordance between the assay results. The Templex assay provided type-specific results and found multiple types in 29.2% (14 of 48) of high-risk HPV-positive samples. The entire Templex procedure, including DNA extraction, can be completed within 5 hours, providing a rapid and reliable diagnostic tool for HPV detection and typing that is amenable to automation.Human papillomavirus (HPV) infection is linked with cervical cancer (1,12,24). HPV can be divided into "high-risk (HR)" and "low-risk" groups on the basis of their association with cervical lesions (7,11,17). The HR group includes HPV types 16,18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 67, 68, 69, 82, 26, 53, 66, 70, and 73, while the low-risk group includes types 6, 11, 40, 42, 43, 44, 54, 61, 72, 81, and 89 (11, 15, 19). Recently, quadrivalent and bivalent vaccines have been demonstrated to effectively prevent type-specific persistent infection and disease (6, 23). To monitor the impact of vaccine implementation strategies, determine type-specific persistence, and evaluate the clinical significance of coinfection with multiple HPV types, HPV testing will require type-specific results. A high-throughput, sensitive, specific, and reproducible HPV detection and typing assay is therefore highly desirable.Most established HPV typing assays used in epidemiologic studies are based on consensus PCR to amplify the relatively conserved L1 gene region with hybridization, restriction enzyme digestion, or sequencing of the amplicon to determine type(s) (2, 22). Widely used L1 consensus primer PCR systems include the GP5ϩ/6ϩ, PGMY09/11, and SPF systems (4, 5, 13, 21). However, in all of these methods, variations in the efficiency of type-specific priming, primer competition, and limitations on the reagent concentrations in the assay may affect the observed type distribution, particularly when large numbers of types at greatly different copy numbers are present. In addition, typing requires a variable number of additional post-PCR steps, such as amplicon purification, gel electrophoresis, hybridization, and washing. These additional steps increase the time and labor required.We report the development of a novel HPV genotyping assay that is based on proprietary Templex technology. Templex is a unique multiplex PCR platform that uses a targetenriched multiple...

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Section: Discussionmentioning

confidence: 99%

Simultaneous Amplification and Identification of 25 Human Papillomavirus Types with Templex Technology

Han

Swan

Smith³

et al. 2006

J Clin Microbiol

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“…Very different HPV detection results were obtained based on the E6 gene detection than when we used consensus primers for L1 detection. The presence of L1 gene PCR products was reported to be found only in 60% of the HPV E6 gene detection system (10). We therefore included the primers for L1 in our system.…”

Section: Discussionmentioning

confidence: 99%

“…PCR was performed for a simultaneous amplification of four HPV16 genes: E1, E2, E6, and L1. We decided to include the detection of L1 gene fragment since PCR methods based on the fact that L1 fragment detection are generally used for confirmation of HPV infection (10).…”

Section: Methodsmentioning

confidence: 99%

Human Papillomavirus Type 16 Status in Cervical Carcinoma Cell DNA Assayed by Multiplex PCR

Łukaszuk¹,

Liss²,

Woźniak³

et al. 2003

J Clin Microbiol

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Integration of human papillomavirus (HPV) DNA into host genome occurs early in cancer development and is probably an important event in malignant transformation of cervical cancer. The HPV genome integration usually disrupts E2 gene open reading frames. It results in the lack of E2 gene suppressor of the synthesis of E6 and E7 products which, in turn, leads to the overexpression of E6 and E7 genes. The oncogenic HPV types (HPV16, -18, -45, and -58) can be present as episomes or may integrate into human chromosomes. Sixty-six cervical cancer patients positive for HPV16 were tested for the presence of E6, E2, E1, and L1 genes. Multiplex PCR was carried out in all cases. Using cluster analysis, the calculated ratios of E1/E6, E2/E6, L1/E6, E1/E2, and E2/(E1*E6) gene amplification products were divided into two or three statistically different groups. These were used for statistical analysis of the prevalence of specific gene types in histological types of cancer, different levels of clinical staging, and histologically confirmed nodal metastases. The statistical analysis proved a significant correlation in the ratios of E2/E6 and E1/E2 only. The E2/E6 and E1/E2 were higher in carcinoma in situ than in advanced squamous cancers. The E2/E6 ratios were lower in higher clinical stages. The multiplex PCR estimation of the E2/E6 ratio could be a simple method for selecting patients with a high risk of a poor outcome in a standard stage-dependent treatment procedure.Human papillomaviruses (HPV) are small (ϳ8 kb) DNA viruses that cause warts and proliferative lesions in epidermal tissues. Infection of the anogenital tract by these viruses is associated with several premalignant and malignant lesions, especially dysplasia and carcinoma of the uterine cervix (18). HPV infection is the most frequent sexually transmitted disease worldwide, and up to 60% of sexually active women will become infected by HPV in the genital tract (19). Even in a healthy population, the incidence of HPV infection varies from ca. 20% in Brazil to ca. 3% in Belgium and Poland (17,25). About 40 types of HPV with affinity to anogenital epithelium are known so far. HPV types 16, 18, 31, and 33 are considered the most important in the process of cervical cancerogenesis (12, 16). Genomic integration of an HPV DNA correlates with increased viral gene expression and cellular growth advantage (8). Integrated forms of HPV genomes are found much more frequently in cervical cancers than in cervical intraepithelial neoplasia cells (9, 14). These observations are consistent with the hypothesis that integration provides a selective advantage to cervical epithelial precursors of cervical carcinoma (8). During a common infection and in most premalignant lesions, HPV is in an episomal state (5). However, most cervical carcinomas and the cell lines derived from them maintain the HPV genome in an integrated form or in both integrated and episomal forms (3, 6). The genome integration of HPV usually disrupts E2 gene open reading frames (6, 9). It results in the lack of E...

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“…Direct comparisons of HPV detection methodology have demonstrated that the sensitivity of our PCR assay is higher than that of PCR assays using MY09 and MY11 and GP17 and GP18 primers. (10,11) A reaction mixture without template DNA was included in every set of PCR runs as a negative control. Also, primers for a fragment of the β-actin gene were used as a control to rule out false-negative results for samples in which HPV DNA was not detected.…”

Section: Methodsmentioning

confidence: 99%

Human papillomavirus infections among Japanese women: age‐related prevalence and type‐specific risk for cervical cancer

et al. 2009

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To obtain baseline data for human papillomavirus (HPV) screening and vaccination in Japan, we analyzed HPV DNA data from 2282 Japanese women ( P ersistent infection with oncogenic human papillomaviruses (HPV), most commonly types 16 and 18, leads to cervical cancer, the second most common cancer in women worldwide.(1) Therefore, oncogenic HPV testing combined with cytology was approved for primary screening in the USA, because of sensitivity and cost-effectiveness.(2) In addition, HPV vaccines have been licensed in the USA, Australia, and European and other countries, because of their efficacy and safety. Clinical studies of HPV vaccines have demonstrated close to 100% protection against HPV16-and HPV18-related infections and diseases, (3)(4)(5) implying possible cross-protection against HPV45, HPV31, and HPV52.(4,5) Based on evidence from clinical trials, (3)(4)(5)(6)(7) these two tools targeting HPV (detection assay and vaccine) are becoming increasingly attractive for cervical cancer prevention worldwide. In Japan, however, HPV DNA testing is still unavailable in mass screening and no HPV vaccine has yet been licensed. Type-specific and age-related data of HPV prevalence, both for women with normal cytology and for women with cervical diseases, are prerequisites to make a well-judged decision about the future role of HPV screening and vaccination in cervical cancer prevention, but these data are missing in Japan. A meta-analysis of Japanese HPV studies provided representative data of HPV type distribution, but no information about age-specific prevalence.In the present study, we analyzed HPV DNA data from 2282 Japanese women to obtain the prevalence data of HPV among women across a broad age range. Our data may help provide models for further evaluating potential impact and cost effectiveness of HPV screening and vaccination in Japan. Materials and MethodsStudy subjects. Our study subjects consisted of 2282 Japanese women (1517 normal HPV detection and genotyping. Exfoliated cells from the ectocervix and endocervix were collected into a tube containing 1 mL PBS and stored at -30°C until DNA extraction. We detected HPV DNA in cervical samples by PCR-based methodology described previously.(9) In brief, total cellular DNA was extracted from cervical samples by a standard sodium dodecyl sulfateproteinase K procedure. HPV DNA was amplified by PCR using consensus primers (L1C1 and L1C2 + L1C2 M) for the HPV L1 region. Direct comparisons of HPV detection methodology have demonstrated that the sensitivity of our PCR assay is higher than that of PCR assays using MY09 and MY11 and GP17 and GP18 primers. (10,11) A reaction mixture without template DNA was included in every set of PCR runs as a negative control. Also, primers for a fragment of the β-actin gene were used as a control to rule out false-negative results for samples in which HPV DNA was not detected. To avoid contamination, we used disposable utensils and discarded them after a single use. We also used aliquoted reagents and maintained separate locations ...

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Detection of Human Papillomaviruses in Cervical Neoplasias Using Multiple Sets of Generic Polymerase Chain Reaction Primers

Cited by 33 publications

References 16 publications

Simultaneous Amplification and Identification of 25 Human Papillomavirus Types with Templex Technology

Simultaneous Amplification and Identification of 25 Human Papillomavirus Types with Templex Technology

Human Papillomavirus Type 16 Status in Cervical Carcinoma Cell DNA Assayed by Multiplex PCR

Human papillomavirus infections among Japanese women: age‐related prevalence and type‐specific risk for cervical cancer

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