3.3 Analytical sensitivity using viral DNA/RNA as template
Combined CaPV and PaPV DNAs, mixed at equal ratio, were serially diluted in negative skin DNA and used as template for the CaP rule-out assays; and combined PaPV DNA and FMDV RNA, mixed at equal ratio, were serially diluted in negative skin DNA and used as template for the FMD rule-out assays. The estimated LODs (highest detectable dilution per assay) calculated from the standard curves (Ct vs. log10template dilutions) were: 106 (LSDV, GPV, SPV and PaPV), by CaP rule-out and the corresponding singleplex assays (Table 1 & Fig 2); 106 (PaPV, FMDV Asia1, C, SAT1 and SAT3), by FMD rule-out and the corresponding singleplex assays (Table 2 & Figure 3), and 107 (FMDV A, O, and SAT2), by FMD rule-out and the corresponding singleplex assays (Table 2 & Figure 3). The LODs, with respect to virus titers (TCID50/ml per assay) were: between 100.3 and 101.6for LSDV, GPV and SPV, by CaP rule-out assays (Table 1), and between 100.45 and 101.8 for FMDV serotypes A, Asia1, C, O, SAT1, SAT2 and SAT3, by FMD rule-out assays (Table 2). The AE and R2 values, calculated from the standard curves (Ct vs. log10 template dilutions) were 94-106% (AE) and >0.99 (R2), respectively, by CaP rule-out assays (Table 1), and 95-105% (AE) and >0.99 (R2), respectively, by FMD rule-out assays (Table 2). The AE and R2 values of the multiplex assays are in close agreement with that of the corresponding singleplex assays (Tables 1 & 2). It should be noted that the LOD (highest detectable dilution per assay) of PaPV by singleplex assays was not shown, which remained same (106) as the corresponding multiplex (CaP and FMD rule-out) assays.
For CaP/FMD rule-out assays, combined LSDV and PaPV DNAs were mixed at equal ratio with FMDV-A, FMDV-C or FMDV-O RNA, and serially diluted in negative skin DNA and used as template. The LOD, AE and R2 values were estimated from the standard curves (Supplemental Fig. 1) and the results are summarized in Table 3. The estimated LOD (highest detectable dilution per assay) was 106 for all viruses including the FMDV serotypes A and O, which had the LODs dropped by 10-fold compared to the corresponding singleplex and CaP or FMD rule-out assays (LOD 107). The CaP/FMD rule-out assays were also carried out with combined GPV and PaPV or SPV and PaPV DNAs, mixed at equal ratio with FMDV-O or FMDV-C RNA and serially diluted in the negative DNA and used as template. The LOD was 106 dilution per assay for all viruses including FMDV-O which was dropped by 10-fold compared to the corresponding singleplex and CaP or FMD rule-out assays (Supplemental Fig. 2). The AE and R2 values of the CaP/FMD rule out assays, calculated from the standard curves, were 96-116% (AE) and >0.98 (R2), respectively, which are very close to their acceptable ranges (AE 90-110%; R2>0.99) (Table 3; Supplemental Figs 1 & 2).