2.4 Multiplex qPCR and RT-qPCR assays
Three multiplex assay designs were tested and optimized: 1) a capripox (CaP) rule-out (3-plex) assay for simultaneous detection and differentiation of CaPV and PaPV, 2) a FMD rule-out (3-plex) assay for simultaneous detection and differentiation of PaPV and FMDV, and 3) a CaP/FMD rule-out (4-plex) assay for simultaneous detection and differentiation of CaPV, PaPV and FMDV. All multiplex assays includedACTB (β actin) as the IPC. The final selection of the reporter/quencher dyes for the TaqMan™ probes were: FAM/MGBNFQ (CaPV), Cy5/IADQ (PaPV) and VIC/QSI (ACTB ) for CaP rule-out assays; FAM/MGBNFQ (FMDV), Cy5/IADQ (PaPV) and VIC/QSI (ACTB ) for FMD rule-out assays; and FAM/MGBNFQ (CaPV), Texas Red/IADQ (FMDV), Cy5/IADQ (PaPV) and VIC/QSI (ACTB ) for FMD/CaP rule-out assays. The composition of the PCR mastermixes and the thermocycling conditions used in the multiplex assays were the same as used in the corresponding singleplex assays (above), except for FMDV where the primers/probe concentrations were doubled.