To isolate a region of L1 targeted by the LA-HPV and the Abbott Real-Time PCR method, conventional PCR for HPV DNA viral amplification of a ~ 450 bp HPV-specific segment from the L1 gene covering nucleotide positions (5722-6162) numbered according to NC001526 HPV-16 reference genome was performed using the forward primer MY09 5′-CGTCCMARRGGAWACTGATC-3′ and the reverse primer MY11 5′-GCMCAGGGWCATAAYAATGG-3′. Five microliters of DNA were added to 15 μL of reaction mix containing 1 × PCR buffer, 0.2 mM dNTPs, 4 mM MgCl2, 0.3 μΜ of each primer, and 2 U/μL of Platinum Taq DNA Polymerase, High fidelity (Invitrogen, USA).
The thermocycling conditions were denaturing at 96 °C for 10 s, annealing at 50 °C for 5 s, and final extension at 60 °C for 4 min for 25 cycles. PCR products were subjected to electrophoresis in 4% agarose (Applichem) using 1 × TBE buffer (Applichem) and visualized under UV light. PCR products were then purified using commercially available SureClean Plus (Bioline) and the purified products were sequenced directly via automated sequencing using two overlapping PCR primers (both forward and reverse). The BigDye Terminator v3.0 kit (Applied Biosystems; Foster City, CA, USA) was used for sequencing using the automated Sequencer (ABI PRISM 3130xl; Applied Biosystems).
The thermocycling conditions were denaturing at 96 °C for 10 s, annealing at 50 °C for 5 s, and final extension at 60 °C for 4 min for 25 cycles. PCR products were subjected to electrophoresis in 4% agarose (Applichem) using 1 × TBE buffer (Applichem) and visualized under UV light. PCR products were then purified using commercially available SureClean Plus (Bioline) and the purified products were sequenced directly via automated sequencing using two overlapping PCR primers (both forward and reverse). The BigDye Terminator v3.0 kit (Applied Biosystems; Foster City, CA, USA) was used for sequencing using the automated Sequencer (ABI PRISM 3130xl; Applied Biosystems).