Abstract
There is a growing appreciation of the potential value for routine screening for the presence of HPV not only for cervical specimens but also from oral cavity. The purpose of this study was to develop and clinically evaluate a single-tube seminested PCR assay for the detection of HPV. Several parameters such as PCR primers, primer annealing temperature, the number of PCR cycles and concentration of PCR components were optimized. The assay was evaluated using HPV inserts of type 6, 11, 16, 18, 31, 33, 38 and 51. Evaluation of seminested PCR assay was performed with cervical scrapings from 30 patients and buccal swabs from 30 head and neck cancer patients and results were compared with those of two-tube nested PCR. The results were found to be comparable with a total of 60% (36/60) of samples being positive for HPV using the single-tube assay, while 62% (37/60) positivity was found with two-tube PCR assay. We succeeded in developing a single-tube seminested PCR method for HPV DNA detection which is easier than the conventional nested PCR and can be further evaluated as a potential screening tool for detecting HPV in oral and cervical regions.
Highlights
Infection with high-risk types of human papillomavirus (HPV) is a well-established risk factor for the development of cervical carcinoma [1]
Association between HPV and the development of certain head and neck cancers has been established recently [2]. This is strengthened by the fact that the oncogenic HPV types detected in cervical carcinomas have been identified in head and neck cancers [3]
MY11/GP6+ polymerase chain reaction (PCR) was performed in 20 l total reaction volume with 100pg of plasmid DNA template using conditions described in previously published literature for MY 09/11 and GP5+/6+ systems [17]
Summary
Infection with high-risk (oncogenic) types of human papillomavirus (HPV) is a well-established risk factor for the development of cervical carcinoma [1]. Association between HPV and the development of certain head and neck cancers has been established recently [2]. This is strengthened by the fact that the oncogenic HPV types detected in cervical carcinomas have been identified in head and neck cancers [3]. Previous studies, including our own work, have found MY/GP two-step nested PCR to be far more sensitive than the USFDA approved Hybrid Capture II assay (Digene), for detecting HPV [6,7,8]. A two-step or two-tube nested PCR is time-consuming, requires two sets of reaction mixtures and is prone to false positive reactions, as transfer from first to second
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