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.