title: Detection of human papillomavirus type-16 DNA utilising microtitre-plate based amplification reactions and a solid-phase enzyme-immunoassay detection system.
abstract: The development of a nested polymerase chain reaction (PCR) assay to detect low concentrations of human papillomavirus type-16 (HPV-16) DNA for epidemiological studies is described. The PCR utilises primers located in the E5 open reading frame, has an analytical sensitivity of 4 HPV-16 genomes and does not produce amplicons from other common genital HPVs (types-6, -11, -18, -31 and 33). This assay was carried out in 96-well plates utilising internal primers labelled with dinitrophenol (DNP) and biotin so that amplicons can be captured onto streptavidincoated plates and detected using an alkaline phosphatase-labelled monoclonal antibody to DNP. The assay was effective for detecting HPV-16 DNA in plasmids, cell-lines and, both freshly collected or archival (formalin-fixed/paraffin embedded) clinical specimens. This system is therefore suitable for epidemiological studies to identify individuals infected with HPV-16 DNA in episomal form who may be at increased risk of developing anogenital carcinomas.
authors.affiliation: Richard Dimbleby Laboratory of Cancer Virology, Department of Virology, Rayne Institute, United Medical School, St Thomas' Hospital, London, UK.
authors.forename: S
authors.initials: S
authors.lastname: Cavuslu
authors.forename: W G
authors.initials: WG
authors.lastname: Starkey
authors.forename: J N
authors.initials: JN
authors.lastname: Kaye
authors.forename: C
authors.initials: C
authors.lastname: Biswas
authors.forename: C
authors.initials: C
authors.lastname: Mant
authors.forename: B
authors.initials: B
authors.lastname: Kell
authors.forename: P
authors.initials: P
authors.lastname: Rice
authors.forename: J M
authors.initials: JM
authors.lastname: Best
authors.forename: J
authors.initials: J
authors.lastname: Cason
mesh_terms: D017931:DNA Primers; D004279:DNA, Viral / Q000032:analysis*; D005069:Evaluation Studies as Topic; D005260:Female; D006367:HeLa Cells; D006801:Humans; D007124:Immunoenzyme Techniques; D009856:Oncogene Proteins, Viral / Q000235:genetics*; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D050725:Papillomavirus E7 Proteins; D016133:Polymerase Chain Reaction / Q000379:methods*; D012097:Repressor Proteins; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured; D002583:Uterine Cervical Neoplasms / Q000473:pathology / Q000821:virology*; D002578:Uterine Cervical Dysplasia / Q000473:pathology / Q000821:virology*; D000086082:Viroporin Proteins
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D012097:Repressor Proteins; C068651:E6 protein, Human papillomavirus type 16; C054859:oncogene protein E5, Human papillomavirus type 16; D000086082:Viroporin Proteins; C059731:oncogene protein E7, Human papillomavirus type 16