title: The use of general primers GP5 and GP6 elongated at their 3' ends with adjacent highly conserved sequences improves human papillomavirus detection by PCR.
abstract: Sequence analysis of human papillomavirus (HPV) general primer GP5/6 mediated PCR products revealed the presence of short highly conserved sequences adjacent to the 3' ends of both primers. Part of these sequences was used to elongate GP5 and GP6 at their 3' ends to generate the primers GP5+ and GP6+, respectively. Compared with the GP5/6 PCR, GP5+/6+ specific PCR on 22 cloned mucosotropic HPVs revealed an improved HPV detection, reflected by a 10- to 100-fold higher sensitivity and a markedly increased signal to background ratio, especially at the gel level. As determined on purified DNA, the sensitivity of this GP5+/6+ based assay was at the femtogram level for those HPV genotypes which match strongly with the primers (e.g. HPV-16) and at the picogram level for HPV types (e.g. HPV-39 and -51) having four or more mismatches with one or both primers. Application of both methods on 264 cervical scrapes of a cohort of women participating in a prospective follow-up study revealed an increase of total HPV positivity from 39% (GP5/6 PCR) to 43% (GP5+/6+ PCR) of the scrapes. Additional HPV typing by PCR specific for the HPV-6, -11, -16, -18, -31 and -33 revealed that all GP5+/6+ PCR positive cases which were negative by GP5/6 PCR (n = 12) contained HPV types different from these six types. These data indicate that the GP5+/6+ PCR method provides an increased detection level mainly of uncommon, apparently poorly matched HPV types in cervical scrapes and most likely in the enlargement of the spectrum of HPVs detectable by this assay.
authors.affiliation: Department of Pathology, Section of Molecular Pathology, Free University Hospital, Amsterdam, The Netherlands.
authors.forename: A M
authors.initials: AM
authors.lastname: de Roda Husman
authors.forename: J M
authors.initials: JM
authors.lastname: Walboomers
authors.forename: A J
authors.initials: AJ
authors.lastname: van den Brule
authors.forename: C J
authors.initials: CJ
authors.lastname: Meijer
authors.forename: P J
authors.initials: PJ
authors.lastname: Snijders
mesh_terms: D017124:Conserved Sequence; D017931:DNA Primers; D004279:DNA, Viral; D006801:Humans; D027383:Papillomaviridae / Q000235:genetics / Q000302:isolation & purification*; D030361:Papillomavirus Infections / Q000821:virology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014412:Tumor Virus Infections / Q000821:virology*
chemical_list: D017931:DNA Primers; D004279:DNA, Viral