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).