2.3 Singleplex qPCR and RT-qPCR assays
All singleplex (one target) assays reported in this study were carried out on ABI 7500 Fast thermocycler (Thermo Fisher Scientific). The OIE recommended real-time PCR assays were used for the detection of CaPV (Balinsky et al. 2008) and FMDV (Callahan et al. 2002), and a recently published qPCR assay (Das et al. 2017a) was used for the detection of PaPV. The beta actin gene ACTB, ubiquitously expressed in all mammalian species, including ruminants (sheep, goat and cow) was used as an internal positive control (IPC), and amplified as described previously (Das et al. 2017b). The original CaPV and FMDV assays were slightly modified by replacing their PCR chemistries as reported earlier (Das et al. 2017b; Wang et al. 2020). The qPCR assays were carried out using Path-ID™ qPCR Mastermix (Thermo Fisher Scientific), and the RT-qPCR assays were carried out using Path-ID™ Multiplex One-Step RT-PCR Kit (Thermo Fisher Scientific). The TaqMan™ probes used in qPCR/RT-qPCR were labeled with FAM (CaPV or PaPV) or VIC (ACTB ) as the reporter dye and TAMRA (FMDV and PaPV) or MGBNFQ (CaPV) as the quencher dye. A complete list of the primers and probes used in the PCR assays are shown in supplemental table 2. The composition of the PCR mastermixes included 1x kit-suppled buffer (mastermix), forward primer (FP), reverse primer (RP), probe (Pr) and 2 µl of template (viral DNA or RNA), plus balance nuclease-free water to a final volume of 25 µl. The concentrations of the primers and probe used in the PCR mastermixes were: 0.25 µM FP, 0.625 µM RP and 0.25 µM Pr for CaPV qPCR; 0.2 µM FP, 0.2 µM RP and 0.2 µM Pr for PaPV orACTB qPCR and 0.2 µM FP, 0.2 µM RP and 0.2 µM Pr for FMDV RT-qPCR. The thermocycling conditions for qPCR (CaPV, PaPV orACTB ) included 1 cycle of 95 ºC for 10 min followed by 45 cycles of 95 ºC for 15 sec and 65 ºC for 1 min. The thermocycling conditions for RT-qPCR (FMDV) included one cycle of 45 ºC 10 min (RT step) and then 1 cycle of 95 ºC for 10 min followed by 45 cycles of 95 ºC for 15 sec and 65 ºC for 1 min.