DETECTION OF HUMAN PAPILLOMAVIRUS TYPE-16 DNA UTILIZING MICROTITRE-PLATE BASED AMPLIFICATION REACTIONS AND A SOLID-PHASE ENZYME-IMMUNOASSAYDETECTION SYSTEM

The development of a nested polymerase chain reaction (PCR) assay to d etect low concentrations of human papillomavirus type-16 (HPV-16) DNA for epidemiological studies is described. The PCR utilises primers loc ated in the E5 open reading frame, has an analytical sensitivity of 4 HPV-16 genomes and does not produce amplicons from other common genita l 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 streptavidin-c oated plates and detected using an alkaline phosphatase-labelled monoc lonal antibody to DNP. The assay was effective for detecting HPV-16 DN A in plasmids, cell-lines and, both freshly collected or archival (for malin-fixed/paraffin embedded) clinical specimens. This system is ther efore suitable for epidemiological studies to identify individuals inf ected with HPV-16 DNA in episomal form who may be at increased risk of developing anogenital carcinomas.