3.2 Analytical sensitivity using PACs as template
The PACs (cloned plasmid or in-vitro transcribed RNA) were serially diluted in nuclease-free water (singleplex) or in negative skin DNA (multiplex) and used as template. In the CaP rule-out assays, the CaPV PAC was co-amplified with ACTB in the presence of primers and probe of PaPV; and in the FMD rule-out assays, the FMDV PAC (transcribed RNA) was co-amplified with ACTB in the presence of primers and probe of PaPV. The PaPV PAC was subjected to two multiplex rule-out assays, PaP1 and PaP2. In PaP1, the PAC was co-amplified with ACTB in the presence of primers and probe of CaPV; and in PaP2, the PAC was co-amplified withACTB in the presence of primers and probe of FMDV. The LODs (copy number/assay), estimated from the standard curves (Ct vs. log10 template dilutions), were 2 (CaPV), 7 (PaPV) and 15 (FMDV) by both singleplex and multiplex assays (Figs 1A, 1B and 1C). The AE and R2 values, calculated from the standard curves were: 96 -110% (AE) and >0.99 (R2), respectively, for singleplex assays; and 94-106% (AE) and >0.99 (R2), respectively, for multiplex assays, regardless of origin of the PACs. These results show linearity of amplification of the PACs by the multiplex assays with minimal interference from the primers and probes of the non-target assays including the ACTB .