Detection and genotyping of human papillomavirus by real-time PCR assay |
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| Authors: | Frédérique Moreau Rachid Fetouchi Isabelle Micalessi Valérie Brejeon Nathalie Bacon Geert Jannes Catherine Le Pendeven Bouchra Lekbaby Dina Kremsdorf Jean Lacau Saint Guily Patrick Soussan |
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| Institution: | 1. Service de Virologie, Hôpital Tenon et Université Pierre et Marie Curie, Paris, France;2. INSERM U845, Université Paris Descartes, Faculté de Médecine René Descartes, Paris, France;3. Applied Molecular Biology Research group, Laboratoire de biologie cellulaire et histologie, Université d’Anvers, Anvers, Belgium;4. Innogenetics NV, Technologiepark 6, B9052 Ghent, Belgium;5. Service d’Oto-Rhino-Laryngologie, Hôpital Tenon et Université Pierre et Marie Curie, Paris, France;1. Department of Molecular Medicine;2. Department of Information Engineering, University of Padova, Padova, Italy;1. Department of Laboratory Medicine, Karolinska Institutet and Karolinska University Hospital, 141 86 Stockholm, Sweden;2. Department of Urology and Andrology, Northwestern State Medical University n.a. I.I. Mechnikov, 191015 St. Petersburg, Russia;1. Hannover Medical School (MHH), Department of Gastroenterology, Hepatology and Endocrinology, Carl-Neuberg-Straße 1, 30623 Hannover, Germany;2. Robert Koch Institute, Department of Infectious Diseases, Seestraße 10, 13353 Berlin, Germany;3. Institute of Tropical Medicine, University of Tuebingen, Wilhelmstraße 27D, 72074 Tübingen, Germany;4. Department of Infectious Diseases and Hepatology, Medical University of Bialystok, ?urawia 14, 15540 Bia?ystok, Poland;5. ifi – Institut für Interdisziplinäre Medizin, Lohmühlenstraße. 5, 20099 Hamburg, Germany;1. Research Center for Bioengineering and Sensing Technology, School of Chemistry and Biological Engineering, University of Science and Technology Beijing, Beijing, 100083, PR China;2. Department of Chemistry, Beijing Key Laboratory of Microanalytical Methods and Instrumentation, MOE Key Laboratory of Bioorganic Phosphorus Chemistry & Chemical Biology, Tsinghua University, Beijing, 100084, PR China;3. State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing, 100029, PR China;4. School of Biomedical Engineering, Shenzhen University, Shenzhen, Guangdong 518060, PR China;1. Scottish Human Papillomavirus Reference Laboratory, Directorate of Laboratory Medicine, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh EH16 4SA, United Kingdom;2. Department of Mathematics and Statistics, University of Strathclyde, Livingstone Tower, 26 Richmond Street, Glasgow G1 1XH, United Kingdom;3. Edinburgh Specialist Virology Centre, Directorate of Laboratory Medicine, Royal Infirmary of Edinburgh, 51 Little France Crescent, Edinburgh EH16 4SA, United Kingdom |
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| Abstract: | BackgroundDiagnosis 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.ObjectiveOur 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 designValidation 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.ResultsData 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.ConclusionThe 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. |
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