*2.2. HR HPV Detection and Genotyping*

The ThinPrep cervical smear samples were stored at 4–8 ◦C for up to 3 days from the sampling day. The 2 mL of collected samples were transferred to nuclease-free tubes and centrifugated at 8000× *g* for 5 min. The formed pellet was dissolved in 200 µL of nuclease-free water and used for nucleic acid extraction. According to the manufacturer's instructions, DNA extraction was carried out using the SaMag STD DNA Extraction Kit (Sacace Biotechnologies, Como, Italy). The extracted DNA was eluted in 100 µL elution buffer. The detection and genotyping of 12 HR HPV genotypes (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, and 59), marked as the HPV DNA test, were performed using the High Risk Typing Real-TM Kit (Sacace Biotechnologies, Como, Italy) following manufacturer's instructions. The E7 gene of specific HPV genotypes was amplified using primers and TaqMan probes in the multiplex reaction performed in a total of 13 µL. The β globin gene is used as an internal control. Real-time PCR was performed on the SaCycler-96 (Sacace Biotechnologies, Como, Italy). After the initial activation of the DNA polymerase at 95 ◦C for 15 min, five cycles of amplification were performed under the following conditions: 95 ◦C/5 s, 60 ◦C/20 s, and 72 ◦C/15 s, and 40 amplifications were performed under the following conditions: 95 ◦C/5 s, 60 ◦C/30 s (fluorescence detection), and 72 ◦C/15 s. The kinetics of the detected fluorescence signals were monitored using the SaCycler-96 software package (Sacace Biotechnologies, Como, Italy).
