Cfx96 real time system of the c1000 touch thermal cycler

Using the real-time qPCR assay, the online web interface from IDT² primers and probes were designed. The amplicon length was set by the program to obtain 70–150 bp within the target regions. The primers and probes used for the detection of the four OaPV genotypes (OaPV1-2-3 and 4) are reported in Table 1. Primers and probes were ordered as a mix with a primer-to-probe ratio of 3.6. The qPCR reaction mixture was prepared by adding 7 μL of template (100 ng genomic DNA), 10 μL of 2X SsoAdvanced™ Universal Probes Supermix (Bio-Rad Laboratories, Hercules, CA, USA), 1 μL of target probe (FAM) /primer mix (final concentration of 900 nM of each primer and 250 nM of probe) in a total volume of 20 μl. DNA quality and concentration were assessed using a Nanodrop (Thermo Scientific, MA, USA). Four separate PCR reactions were performed using the CFX96 Real-Time System of the C1000 Touch^TM Thermal Cycler (Bio-Rad Laboratories, Hercules, CA, USA). The thermal cycling conditions were as follows: 50°C for 2 min, 95°C for 10 min, and 40 cycles of 95°C for 15 s and 58°C for 60 s. Each sample was analyzed in duplicate, and negative controls were included in all runs. Data acquisition and analysis were performed using the CFX Maestro™ (Bio-Rad Laboratories, Hercules, CA, USA) software. The same samples used as positive controls for ddPCR were also tested using qPCR.