HPV typing
Total DNA isolated from formalin-fixed tissue blocks was used for HPV typing. Real-time PCR using Sybr®Green and specific primers for HPV16, 18, and 33, was performed on a 7900HT Fast Real-Time PCR System (Applied Biosystems, Foster City, CA). Multiplexed amplification was done in a 25 µl volume using SYBR Green PCR Master Mix at the final concentration 1X, HPV16 primers at 0.7 µM each, HPV18 and 33 primers at 1 µM each, DNA template (up to 100 ng) and nuclease-free water. HPV negative status was confirmed by PCR using consensus GP5+/GP6+ primers, which can detect a large spectrum of HPV types