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).