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.