a LightCycler® 96 of Roche
b CFX96 of Bio-Rad
analytical sensitivity by an order of magnitude than the OIE-recommended qPCR (Figure 2C), indicating a better ability of MADCAT assay to detect virus in clinical samples.
The reproducibility of this method was determined by their intra-assay variability and inter-assay variability using high (5 × 104 copies/μL), medium (5 × 102copies/μL) and low (0.5 copies/μL) concentrations of plasmid DNA. Six independent runs on two real-time quantitative detection systems (CFX96 of Bio-Rad and LightCycler® 96 of Roche) were acquired with each sample tested in quadruplicates on every run. Both intra-assay variability (C.V of Ct value) and inter-assay variability was < 3%, showing reproducible detection and good precision at different viral loads (Table 1).
The specificity of MADCAT was evaluated by testing closely related classical swine fever virus (CSFV), as well as other pathogens of swine: porcine parvovirus (PPV), porcine circovirus type 2 (PCV2), porcine circovirus type 3 (PCV3), pseudorabies virus (PRV), porcine reproductive and respiratory syndrome virus (PRRSV), and Japanese encephalitis virus (JEV). The clinical sample was obtained from previously confirmed infected donor pigs with above-mentioned virus. Fluorescence signal was observed exclusively in ASFV-positive control (Figure 2D).