2.4. Reference Real-time PCR
The OIE-recommended qPCR method (King et al., 2003; OIE, 2018) for ASF
diagnosis was used as the reference technique in comparative assays.
Briefly, genomic DNA was extracted from 200 μL clinical sample (blood,
serum and tissue homogenates) with the TIANamp Genomic DNA Kit (QIAGEN),
and 5 μL of genomic DNA was amplified in a 25 μL reaction, consisting of
0.2 µM of each primer (VP-UP and VP-DP), 0.1 µM of Taqman probe (VP-FP),
and 12.5 μL of 2 × probe qPCR premix (Takara). The thermocycling
parameters and positive judgment criteria are the same as the MADCAT
method.