qPCR
The primers and probes used for the real-time qPCR assay are listed in Table 1. The qPCR reaction mixture (total volume of 20 µL) was prepared by adding 7 µL of template (100 ng genomic DNA), 10 µL of 2X SsoAdvancedTM Universal Probes Supermix (Bio-Rad Laboratories, Hercules, CA, USA), and 1 µL of target probe (FAM)/primer mix. The DNA quality and concentration were evaluated using a NanoDrop spectrophotometer (Thermo Scientific, MA, USA). Four separate PCR were performed using the CFX96 Real-Time System of the C1000 TouchTM Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). The thermal cycling conditions were as follows: 95°C for 5 min, and 40 cycles of 95°C for 15 s and 58°C for 30 s. Each sample was analyzed in duplicate, and positive as well as negative controls were included in all runs. Data acquisition and analysis were performed using CFX MaestroTM (BioRad Laboratories, Hercules, CA, USA) software.