Group-specific differentiation between high- and low-risk human papillomavirus genotypes by general primer-mediated PCR and two cocktails of oligonucleotide probes

Abstract

In recent years, general primer-mediated PCR assays have been developed to detect a broad spectrum of human papillomavirus (HPV) genotypes. In this study, a procedure enabling a simple group-specific differentiation of high-risk (HPV-16,-18,-31,-33,-35,-39,-45,-51,-52,-54,-56, and-58) and low-risk (HPV-6,-11,-34,-40,-42,-43, and 44) HPVs following an HPV general primer-mediated (GP5؉/GP6؉) PCR is presented. By computer-assisted sequence analysis, oligonucleotides (30-mers) specific for 19 different HPV genotypes were selected from the internal part of the 150-bp GP5؉/GP6؉-amplified region. These oligo probes were tested for specificity in a Southern blot analysis of PCR products derived from the same panel of HPV types. No cross-hybridizations were found. The sensitivities of the oligo probes varied from the femtogram level for the well-amplified HPV types like HPV-16 and-18 to the picogram level for the less-well-amplified HPV types like HPV-39 and-51. These sensitivities were reached when the oligo probes were applied both individually and in a cocktail. On the basis of these results, two cocktail oligo probes that enabled a specific and sensitive differentiation between low-and high-risk HPV types were composed.

Isolated human papillomavirus 18-positive extragenital bowenoid papulosis and idiopathic CD4+ lymphocytopenia

“…Hybridization was carried out using a cocktail of digoxigenin-labelled q 2001 British Association of Dermatologists HPV-specific probes, one marking high-risk, and one low-risk HPV types. 10 This identified a high-risk HPV type in the biopsy. The gel was stripped and reprobed with individual HPV type-specific probes, which proved positive for HPV 18.…”

“…The first, using a general primer set known as GP51 and GP61, amplifies a broad spectrum of genital HPV types. 10 The PCR products were electrophoresed on agarose gel and analysed by Southern blotting. Hybridization was carried out using a cocktail of digoxigenin-labelled q 2001 British Association of Dermatologists HPV-specific probes, one marking high-risk, and one low-risk HPV types.…”

Human papilloma virus in the sperm cryobank: an emerging problem?

“…DNA extraction from sperm pellet (containing at least 1.0 · 10 6 spermatozoa) was performed by QIAamp DNA Mini Kit (Qiagen, Milan, Italy). The presence of HPV DNA sequences was investigated by nested polymerase chain reaction (PCR) using MY09 ⁄ MY11 as outer primers (Bauer et al, 1991) and GP5+ ⁄ GP6+ as inner primers (Jacobs et al, 1995). HPV-DNA-positive samples were submitted to HPV genotyping by using the Linear Array HPV Genotyping test (Roche Diagnostic, Milan, Italy) able to detect 37 genotypes (all high and intermediate risk and more frequent among low-risk genotypes).…”

The clinical importance of the nomenclature, evolution and taxonomy of human papillomaviruses

“…However, cytology can now be complemented with powerful procedures that detect HPV DNA, and epidemiological studies are defining criteria where these molecular techniques may enter standard gynecological practice. Some of the emerging commercial tests have their roots in efforts to amplify HPV genomes selectively by the polymerase chain reaction (PCR) with "consensus primers", together with oligonucleotides complementary to a diverse range of HPV types and which were originally designed to study the genomic diversity of HPVs Jacobs et al, 1995). As HPV types that cause genital neoplasia were always central to the search for new HPV types, these techniques were biased to detect alpha-papillomaviruses.…”