Droplet-digital PCR (ddPCR)
The ddPCR workflow was executed as reported previously(Hindson et al., 2011; Pinheiro et al., 2012). Briefly, 20 µL reaction mixtures (supermix, primers, and template) were loaded and partitioned into a maximum of 20,000 droplets in the generator cartridge (Bio-Rad, USA). Then, these droplets were transferred to a 96-well PCR plate and placed on a thermal cycler (Bio-Rad) for amplification. The reaction was carried out at 95 °C for 10 min, followed by 40 cycles of 95 °C for 30 s, 60 °C for 1 min, and an extension at 98 °C for 10 min. The reaction was held at 4 °C. Subsequently, the droplets were passed through the QX200 reader (Bio-Rad) and were assigned as positive or negative based on the fluorescence signal, which is used to determine the presence of target DNA. Data were analyzed using QuantaSoft version 1.3.2.0 software (Bio-Rad). Primers and probes are shown in Fig. 3.