Magnapure platform total nucleic acid isolation kit

Swabs were stored at −20°C until analyses. DNA was extracted using the MagnaPure platform (Total Nucleic Acid Isolation Kit, Roche, the Netherlands) and eluted in 100-microliter elution buffer. HPV-DNA was amplified using the SPF₁₀ primer set. Subsequently, HPV-specific amplicons were detected using the DNA enzyme-linked immunoassay (HPV-DEIA, DDL Diagnostics Laboratory, the Netherlands). Amplicons of positive samples were genotyped with the Line probe assay (HPV-LiPA₂₅, DDL Diagnostics Laboratory, the Netherlands), which is able to detect the 12 hrHPV types 16/18/31/33/35/39/45/51/52/56/58/59 [20 (link)].
Serum samples were stored at −80°C until analyses [21 (link)]. HPV antibodies against L1 virus-like particles for types 16 and 18 were assessed using a multiplex immunoassay. Cut-off levels for seropositivity were 9 Luminex Units (LU)/mL for HPV-16 and 13 LU/mL for HPV-18 [22 (link)].

All swabs were stored at −20°C until processing. After thawing and vortexing, 200 µL of the material was used for DNA extraction using the MagnaPure platform (Total Nucleic Acid Isolation Kit, Roche, the Netherlands). Total DNA was eluted in 100 µL elution buffer and 10 µL was used to amplify HPV-DNA with the SPF₁₀ primer set. HPV-specific amplicons were detected using ELISA (HPV-DEIA, DDL Diagnostic Laboratory, the Netherlands). Positive samples were subsequently genotyped with the Line probe assay (HPV-LiPA, DDL Diagnostic Laboratory), which is able to identify 25 genotypes (6, 11, 16, 18, 31, 33–35, 39, 40, 42–45, 51–54, 56, 58, 59, 66, 68, 70, 74).