title: A non-radioactive PCR enzyme-immunoassay enables a rapid identification of HPV 16 and 18 in cervical scrapes after GP5+/6+ PCR.
abstract: In previous studies, general primer mediated PCR (GP5+/6+ PCR) was applied successfully to detect a broad spectrum of human papillomaviruses (HPV) in cervical scrapes. In order to facilitate PCR based HPV detection and typing, a colourimetric microtitre plate based hybridisation assay was developed. The method utilised one biotinylated primer (bio-GP6+) in the GP-PCR. Biotinylated PCR products were captured on streptavidin coated microtitre plates, denaturated and hybridised to digoxigenin (DIG) labelled HPV specific internal oligo probes. The DIG labelled hybrids were detected using an enzyme immunoassay (EIA). Since HPV 16 and 18 are the most common HPV types found in cervical carcinomas, this approach was initiated for these two types. Cross-hybridisation reactions were not detected when the specificity of this PCR-EIA for HPV 16 and 18 was tested on a panel of 20 different HPV genotypes. The sensitivity of the assay was found to be between 10 and 100 HPV 16 and 18 viral genomes in a background of 100 ng cellular DNA. This was similar to the detection limit of Southern blot analysis of PCR products with radioactively labelled oligonucleotides. A group of cytomorphologically normal (n = 89) and abnormal (n = 96) cervical scrapes were composed of HPV 16 and HPV 18 positive and HPV negative scrapes. All HPV 16 and 18 positive smears were detected by PCR-EIA. These results indicate that PCR-EIA has the potential for a rapid and sensitive HPV DNA test for day-to-day routine examination of cervical scrapes.
authors.affiliation: Department of Pathology, Academic Hospital Free University, Amsterdam, The Netherlands.
authors.forename: M V
authors.initials: MV
authors.lastname: Jacobs
authors.forename: A J
authors.initials: AJ
authors.lastname: van den Brule
authors.forename: P J
authors.initials: PJ
authors.lastname: Snijders
authors.forename: T J
authors.initials: TJ
authors.lastname: Helmerhorst
authors.forename: C J
authors.initials: CJ
authors.lastname: Meijer
authors.forename: J M
authors.initials: JM
authors.lastname: Walboomers
mesh_terms: D000914:Antibodies, Viral / Q000032:analysis*; D015139:Blotting, Southern; D004279:DNA, Viral / Q000032:analysis*; D005260:Female; D006801:Humans; D007124:Immunoenzyme Techniques; D007553:Isotope Labeling; D015345:Oligonucleotide Probes; D027383:Papillomaviridae / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000821:virology*; D014626:Vaginal Smears
chemical_list: D000914:Antibodies, Viral; D004279:DNA, Viral; D015345:Oligonucleotide Probes