3.1 Optimization of the multiplex assays
The original CaPV, PaPV and FMDV assays (above) used in the study were optimized and validated on low throughput PCR platform Cepheid SmartCycler (Sunnyvale, CA), which can run a maximum of up to sixteen reactions for each machine run. In this study, the above assays were further optimized on the high throughput PCR platform ABI 7500 Fast which can run a maximum of 96 reactions per run. For performance evaluation, parallel singleplex assays were performed on SmartCycler and ABI 7500 Fast using serial dilutions of viral DNA/RNA as template. The estimated LOD (highest detectable dilution) of each target (CaPV, PaPV or FMDV) remained the same between the two PCR platforms (results not shown).
Several reporter and quencher dyes (fluorophores) were tested for optimization of the multiplex assays and the best results were obtained using FAM, Cy5, Texas Red and VIC as reporter dyes and MGBNFQ (minor groove binding non fluorescent quencher, Thermo Fisher Scientific), IBDQ (Iowa Black® dark quencher, IDT) and QSY (Thermo Fisher Scientific) as quencher dyes. The quencher dye TAMRA, used in the original singleplex assays (PaPV, FMDV, and ACTB ), was found unsuitable for multiplex assays as it interfered with the fluorescence spectra of other reporter dyes resulting in inconsistent Ct values. Therefore, TAMRA was replaced with other quencher dyes (above). Optimized performance of the multiplex assays were obtained using FAM (CaPV or FMDV), Cy5 (PaPV) and VIC (ACTB ) as reporter dyes for CaP and FMD rule-out assays, and FAM (CaPV), Cy5 (PaPV), Texas Red (FMDV) and VIC (ACTB ) for CaP/FMD rule-out assays (see results below).