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).