3.2. Laboratory Validation of the MADCAT Assay
To systematically ascertain the diagnostic performance of MADCAT assay
for ASFV detection, we used plasmids and clinical whole blood sample to
assess the sensitivity, reproducibility and specificity of this assay.
A standard curve was constructed with a significant linear relationship
(R2=0.9980) and a linear dynamic range across seven
orders of magnitude. The limit of detection (LOD) was 0.5 copies/μL of
DNA sample (Figure 2A, B). There was no statistical difference (P> 0.05) of Ct values between the MADCAT, which underwent
capture before real-time PCR amplification, and standard real-time PCR
(Figure 2B), indicating that all the target DNA released after lysis can
be captured without any loss. When we spiked plasmid DNA of the same
series of concentrations in porcine blood and tested with our method,
the same LOD and linear dynamic range were observed. For the clinical
whole blood sample, a 10-fold dilutions series were tested with MADCAT.
In comparison, the DNAs extracted from the same blood samples were
tested with the OIE-recommended qPCR. The results showed that the MADCAT
method had increased
Table 1. Intra- and inter-assay variability of this method.