title: Detection and genotyping of human papillomavirus by real-time PCR assay.
abstract: 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.
  
  
  METHODS
  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/10(3)cells using in-house HPV (6, 11 and 16) viral load assays.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Service de Virologie, Hôpital Tenon et Université Pierre et Marie Curie, Paris, France.
authors.forename: Frédérique
authors.initials: F
authors.lastname: Moreau
authors.forename: Rachid
authors.initials: R
authors.lastname: Fetouchi
authors.forename: Isabelle
authors.initials: I
authors.lastname: Micalessi
authors.forename: Valérie
authors.initials: V
authors.lastname: Brejeon
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bacon
authors.forename: Geert
authors.initials: G
authors.lastname: Jannes
authors.forename: Catherine
authors.initials: C
authors.lastname: Le Pendeven
authors.forename: Bouchra
authors.initials: B
authors.lastname: Lekbaby
authors.forename: Dina
authors.initials: D
authors.lastname: Kremsdorf
authors.forename: Jean
authors.initials: J
authors.lastname: Lacau Saint Guily
authors.forename: Patrick
authors.initials: P
authors.lastname: Soussan
mesh_terms: D001003:Anal Canal / Q000821:virology; D052160:Benzothiazoles; D003365:Costs and Cost Analysis; D017931:DNA Primers / Q000235:genetics; D003959:Diamines; D005838:Genotype; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000191:economics / Q000379:methods*; D015345:Oligonucleotide Probes / Q000235:genetics; D009930:Organic Chemicals / Q000378:metabolism; D009960:Oropharynx / Q000821:virology; D027383:Papillomaviridae / Q000145:classification* / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000175:diagnosis* / Q000821:virology*; D011804:Quinolines; D060888:Real-Time Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D012680:Sensitivity and Specificity; D012867:Skin / Q000821:virology; D013194:Staining and Labeling / Q000379:methods; D014773:Virology / Q000191:economics / Q000379:methods*
chemical_list: D052160:Benzothiazoles; D017931:DNA Primers; D003959:Diamines; D015345:Oligonucleotide Probes; D009930:Organic Chemicals; D011804:Quinolines; C098022:SYBR Green I