Fig. 4
Analytical sensitivity of HRM assay. a Aligned Melt Curves of different concentrations of the plasmids. The two plasmids, diluted from 4.2 × 100 to 4.2 × 108 copies/μl, were amplified by qPCR and analyzed by HRM analysis software. The results showed FPV and CPV were successful distinguished in the dilution range. Standard curves obtained for FPV (b) and CPV (c) indicating the linearity and efficiency for detecting both viruses by qPCR. The dilutions of standard DNA are indicated on the x-axis, whereas the corresponding cycle threshold (Ct) values are presented on the y-axis. The assays were linear in the range of 4.2 × 100 to 4.2 × 108 template copies/μl, with a coefficient of determination (R2) of 0.999 for both FPV and CPV and reaction efficiency of 97.76 and 95.24%. The result showed that the limit detection of HRM assay is both 4.2 copies/μl for FPV and CPV