Bead-based multiplex genotyping of 58 cutaneous human papillomavirus types

Abstract

Cutaneous human papillomaviruses (HPVs) are a heterogeneous, nonmonophyletic assembly, comprising about 50 characterized types and at least 133 isolates putatively representing new types. Their natural history of infection and potential association with nonmelanoma skin cancer are not well understood. Several PCR systems have been developed that amplify a broad spectrum of cutaneous HPVs. However, amplicon genotyping by sequencing or reverse line blot assays are complex and not well suited for high-throughput analyses. We developed a novel multiplex cutaneous papillomavirus genotyping (McPG) assay for 38 defined and 20 putative cutaneous HPVs of the beta, gamma, mu, and nu genera. Viral DNA was amplified by the use of a modified single-tube nested "hanging-droplet" FAP PCR. The amplifiable papillomavirus (PV) spectrum was enlarged by the use of 9 outer and 13 inner primers. Biotinylated PCR products were hybridized to type-specific oligonucleotide probes coupled to fluorescence-labeled polystyrene beads and analyzed using Luminex technology. Analytical sensitivity was analyzed for 38 defined HPVs and was ≤100 genome copies for all types. Integrated β-globin primers allow for simultaneous DNA quality control. McPG is characterized by high reproducibility (κ= 0.84, 95% confidence interval = 0.79 to 0.88), good concordance with the original nested FAP PCR, followed by sequencing (70.2% complete or partial agreement) when 322 skin biopsy DNA samples were analyzed, and improved ability to detect multiple infections (on average 2.5 HPV types per HPV-positive sample compared to 1.7 HPV types with nested FAP-PCR). In conclusion, McPG is a powerful tool for genotyping multiple cutaneous HPVs in a high-throughput format and is thus suitable for large-scale epidemiological studies.

Fig. 1.

Analytical sensitivity of integrated β-globin PCR. McPG was performed using a 10-fold dilution series of HP DNA. PCR was performed on 10 individually prepared dilution series. The net MFI values obtained after hybridization of the PCR products are shown. The mean value is indicated by solid lines, and the cutoff is indicated by a dashed line.

Fig. 2.

Detection of multiple infections. McPG was performed using a 10-fold dilution series of single HPV types and mixtures comprising 5, 10, 15, and 20 HPV types in 100 ng of HP DNA/μl. Net MFI values obtained after hybridization of the PCR products are shown. The cutoff is indicated by horizontal dashed line.

Fig. 3.

Frequency of detection of different HPV and FA types by McPG and conventional nFAP in 322 skin biopsyspecimens (for HPV or FA types with more than four positive samples by either McPG or nFAP).