Clinical specimens HPV genotyping by the 18 HR HPV MALDI-TOF MS assay

The collected samples in PreservCyt medium for routine liquid-based cytology (LBC) were processed in a room of the laboratory physically separated from where the PCR amplification was performed. Briefly, 2 ml of cellular liquid in PreservCyt medium was removed into an Eppendorf tube for sedimentation. Then the gravity-sedimentary cellular material was lysed in a 400 μl of digestion solution containing 5ul of proteinase at 65 °C for 30 min, followed by treatment in 150 μl NaCl solution. The DNA in the lysed supernatant was precipitated by water-free alcohol, and the pellets were dissolved in TE buffer. The clinical DNA specimens were detected and genotyped using the developed 18 HR HPV MALDI-TOF MS assay. To avoid cross-contamination, the laboratory spaces were separated into three parts: the rooms for pre-PCR processes (DNA extraction, quantification and gel electrophoresis), for PCR processes (PCR reaction system preparation and reactions), and for post-PCR processes (PCR product extensions and conducts of mass spectrometry), respectively.