Title
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Detection and genotyping of human papillomavirus by real-time PCR assay
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Author
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Abstract
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Background: Diagnosis of human papillomavirus (HPV) disease remains a challenge due to several factors related to the cost, the workload of available commercial assays to detect and genotype HPV, and to the low prevalence of infected patients. Objective: Our study aimed to develop a real-time PCR, based on SPF10 primers, in order to combine HPV-DNA detection and genotype identification avoiding the negative samples. Study design: Validation of SYBR-green based SPF10 real-time PCR on HPV-DNA plasmids followed by the investigation of the viral status in 92 samples from oropharyngeal (94%) cutaneous biopsies (3%) and anal smears (3%) which had previously been HPV-genotyped by LiPA hybridization. In-house HPV viral loads were performed to evaluate the SPF10 real-time PCR sensitivity. Results: Data showed that 100% of HPV plasmids, assessable by LiPA hybridization, were detected and genotyped appropriately after SPF10 real-time PCR assays. These results defined a range of melting temperature peaks for HPV positivity by real-time PCR. The efficient determination of the presence of HPV-DNA by SPF10 real-time PCR was validated for 98% of clinical samples compared to commercial method. Discordant results were due to a low HPV-DNA amount and to a supplementary HPV genotype identified. The SPF10 real-time PCR sensitivity was evaluated between 1 and 10 copies/103 cells using in-house HPV (6, 11 and 16) viral load assays. Conclusion: The real-time PCR method was efficient in combining screening and genotyping of HPV-DNA. Cost and workload reduction by SPF10 real-time PCR approach may facilitate earlier diagnosis and clinical management of HPV infected patients. (C) 2012 Elsevier B. V. All rights reserved. |
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Language
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English
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Source (journal)
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Journal of clinical virology. - Rome
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Publication
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Rome
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2013
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ISSN
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1386-6532
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DOI
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10.1016/J.JCV.2012.11.003
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Volume/pages
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56
:3
(2013)
, p. 244-249
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ISI
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000314022500011
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Full text (Publisher's DOI)
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Full text (publisher's version - intranet only)
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