Use of the polymerase chain reaction to specifically amplify integrated HPV-16 DNA by virtue of its linkage to interspersed repetitive DNA

Objective Human papillomavirus (HPV) has been identified as the principal etiologic agent for cervical cancer and its precursors. Different HPV types have been associated with different oncogenic potential. The purpose of this study was to evaluate the relationship between specific HPV type infection and expression pattern of the ras family oncogenes in different grades of HPV-associated human cervical neoplasia. Methods HPV typing was performed using polymerase chain reaction (PCR) in 31 HPV-positive human cervical specimens from patients with squamous intraepithelial lesions (SIL) or squamous cervical carcinoma (SCC). The mRNA expression levels of H-, K- and N-ras oncogenes were examined using the reverse transcriptase polymerase chain reaction (RT-PCR) technique. Statistical analyses were performed using SPSS software. Results Among patients with SCC, H-, K- and N-ras expression levels were higher in HPV 16/18-associated cases compared to HPV 16/18-unassociated samples (p=0.003, p=0.004 and p=0.0001, respectively). The expression levels for H-, K-and N-ras were significantly higher in SCC patients with multiple HPV infection compared with SCC patients with single HPV infection (p=0.009, p=0.01 and p=0.021, respectively). Among patients with SIL, no statistically significant relationship was found between ras expression and HPV status. Conclusion Our findings indicate the possible role of ras signaling interaction with “high-risk” HPV 16/18 and multiple HPV infection in cervical cancer development.

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Genotyping human and bovine isolates ofCryptosporidium parvumby polymerase chain reaction-restriction fragment length polymorphism analysis of a repetitive DNA sequence

FEMS Microbiology Letters ◽

10.1111/j.1574-6968.1996.tb08107.x ◽

1996 ◽

Vol 137 (2-3) ◽

pp. 207-211 ◽

Cited By ~ 67

Author(s):

Alain Bonnin ◽

Marie Noelle Fourmaux ◽

Jean FranÃ§ois Dubremetz ◽

Richard Gordon Nelson ◽

Philippe Gobet ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Restriction Fragment Length Polymorphism ◽

Dna Sequence ◽

Repetitive Dna ◽

Length Polymorphism ◽

Fragment Length Polymorphism ◽

Polymorphism Analysis ◽

Chain Reaction ◽

Polymerase Chain ◽

Restriction Fragment Length

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Detection of Human Papillomavirus in Cervical Gradings by Immunohistochemistry and Typing of HPV 16 and 18 in High-Grades by Polymerase Chain Reaction

Journal of Laboratory Physicians ◽

10.4103/0974-2727.66711 ◽

2010 ◽

Vol 2 (01) ◽

pp. 031-036 ◽

Cited By ~ 3

Author(s):

Mrudula Soma ◽

Suhasini Kamaraj

Keyword(s):

Polymerase Chain Reaction ◽

Human Papillomavirus ◽

High Risk ◽

Tamil Nadu ◽

Low Grade ◽

Chain Reaction ◽

Hpv 16 ◽

Screening Programs ◽

South Indian ◽

Polymerase Chain

ABSTRACT Background: Cervical cancer has a major impact on developing countries, where screening programs are not well established or effective. Aim: This study aims to investigate Human papillomavirus (HPV) 6, 11 and 18 expression in cervical biopsies by immunohistochemistry, (IHC) followed by typing of high-risk HPV 16 and 18 in high-grades by polymerase chain reaction (PCR). Settings and Design: During the study period of six months, 30 biopsy samples were obtained from patients attending various gynecology clinics in and around Trichy District, Tamil Nadu, between January and June 2009. Materials and Methods: The ecto- and endoscopic biopsy specimens of the cervix were fixed in 10% buffered formalin; routine paraffin sections were taken for processing and stained with hematoxylin and eosin. The samples were graded as Normal cervicitis, Cervical intraepithelial neoplasia (CIN) I, II, III, and squamous cell carcinoma (SCC), for original diagnosis by pathologists. The extra sections were studied for the expression of HPV 6, 11 and 18 by immunohistochemistry and HPV DNA 16 and18 by PCR. Results: Out of thirty samples, 15 expressed positive and 15 negative for HPV marker. Twenty-seven cases of cervical gradings have been categorized into high grade CIN II/III, SCC (23) and low grade CIN I (4). The high grades were subjected to PCR for high-risk typing. The results revealed that 15 cases were positive for HPV genotype 16 and eight cases for HPV genotype 18. The prevalence of HPV infection was found to be higher in women aged between 50 and 59. Conclusion: This study reveals a significant detection of HPV in the South Indian suspected individuals, by the use of advanced techniques such as IHC and PCR.

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Human papillomavirus genotyping by a polymerase chain reaction-based genechip method in cervical carcinoma treated with neoadjuvant chemotherapy plus radical surgery

International Journal of Gynecological Cancer ◽

10.1136/ijgc-00009577-200407000-00012 ◽

2004 ◽

Vol 14 (4) ◽

pp. 639-649 ◽

Cited By ~ 4

Author(s):

H. J. Huang ◽

S. L. Huang ◽

C. Y. Lin ◽

R. W. Lin ◽

F. Y. Chao ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Human Papillomavirus ◽

Cervical Carcinoma ◽

Radical Surgery ◽

Chain Reaction ◽

Hpv 16 ◽

Hpv Dna ◽

Hpv Genotyping ◽

Polymerase Chain ◽

Hpv 18

The aim of this study was to evaluate the accuracy of human papillomavirus (HPV) genotyping by a polymerase chain reaction (PCR)-based genechip method and to determine the prognostic value of HPV genotype in bulky stage IB or IIA cervical carcinoma treated with neoadjuvant chemotherapy (NAC) and radical surgery. A total of 149 patients had adequate tissue for the study. The SPF1/GP6+ primers were used to amplify a 184 bp fragment. The amplimers were submitted for direct sequencing and hybridization with a genechip using revert-blot detection of 39 types of HPV DNA in a single reaction. Two runs of PCR with respective hybridization were performed for each tumor. The complete concordance of HPV genotyping was 80.5% (120/149) of the paired genechip results. The kappa coefficient was 0.634 (P < 0.0001). HPV DNA sequences were detected in 100% of the specimens, among which 67.8% harbored single type and 32.2% contained multiple types. HPV-16 was detected in 98.7%, HPV-18 in 22.8%, HPV-31 in 0.7%, HPV-45 in 1.3%, HPV-52 in 2.0%, HPV-58 in 6.7%, HPV-59 in 4.7%, and HPV-67 in 0.7%. In multivariate analyses, the HPV genotype [HPV-18 or HPV-16 and HPV-18 only versus all others: relative risk (RR), 2.33; 95% CI, 1.17–4.64; P = 0.016] and pre-NAC tumor size (>5 versus ≤5 cm: RR, 2.25; 95% CI, 1.13–4.48; P = 0.021) were significantly related to overall survival. This PCR-based genechip method is sensitive and reproducible for HPV genotyping. The association of HPV-18 or HPV-16 and HPV-18 with poor outcome in cervical carcinoma treated with NAC plus radical surgery is confirmed.

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Detection of HPV-16 in cell lines and cervical lavage specimens by a polymerase chain reaction-enzyme immunoassay assay

Journal of Medical Virology ◽

10.1002/jmv.1890370105 ◽

1992 ◽

Vol 37 (1) ◽

pp. 22-29 ◽

Cited By ~ 6

Author(s):

François Coutlée ◽

Linda Bobo ◽

Hawwari Abbass ◽

Gina Dalabetta ◽

Ned E. Hook ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Enzyme Immunoassay ◽

Cell Lines ◽

Chain Reaction ◽

Hpv 16 ◽

Polymerase Chain

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Molecular Methods for a Correct Diagnosis of Multiple HPV Infections and Clinical Implications for Vaccine

International Journal of Gynecological Cancer ◽

10.1097/igc.0b013e31820f5eed ◽

2011 ◽

Vol 21 (3) ◽

pp. 545-550 ◽

Cited By ~ 4

Author(s):

Andrea Tinelli ◽

Giuseppe Leo ◽

Domenico Dell'Edera ◽

Fabio Storelli ◽

Maria Maddalena Galante ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Human Papillomavirus ◽

Real Time ◽

Sequence Homology ◽

Direct Sequencing ◽

Chain Reaction ◽

Hpv 16 ◽

Hpv Genotyping ◽

Polymerase Chain ◽

Hpv 18

IntroductionThe human papillomavirus (HPV) family is characterized by minimal genotypic differences corresponding to different virus types. The aim of this study was to detect the HPV coinfections and the inner genotype in a series of 336 cervical-vaginal samples.MethodsA total of 336 cervical-vaginal samples were taken from 2007 to 2009 using specific molecular techniques such as molecular sequencing and hybridizations. The genome amplification of the L1 open reading frame was analyzed by real-time polymerase chain reaction; direct sequencing was performed by SYBR green fluorescent molecule and degenerate primers MY09 and MY11. The HPV genotyping was accomplished via oligonucleotide probe hybridization. The phylogenetic correlations in coinfections were analyzed by sequence homology of the L1 genomic region. Identified genotypes were then compared.ResultsHuman papillomavirus positivity was observed in 125 cases (37.2%), with 21 cases (16.8%) of HPV presence in coinfections. Coinfections involved HPV 16 genotype (8 cases) and HPV 18 (5 cases). The HPV 16 infection was mainly associated with genotypes with a lower-than-broad sequence homology, so the HPV 18 was linked to genotypes represented in the opposite phylogenetic tree.ConclusionsThe combined and steady use of diagnostic procedures, such as real-time polymerase chain reaction, molecular hybridization, direct sequencing, and HPV genotyping test, allow accurate diagnosis of monoinfections and coinfections. This may faciliate the development of specific viral tests and prophylactic anti-HPV vaccines.

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