2.3. Development and optimization of the VNUA-54 real-time PCR assay
In order to design an ASFV p54 –based real-time PCR, the E183L (p54) gene sequences representing all 24 ASF p72 genotypes of ASFV were aligned by the Geneious software, and a highly conserved 100 bp region between nucleotide positions 287 and 386 was selected, and primers (Forward: 5’-CAAGTGTAGGCAAGCCAGTC-3’ and Reverse: 5’-GCCATGACTAGTCTGTCCGT-3’) and a TaqMan® probe (5’-FAM ACGGGCAGACCGGCAACAAA-3’TAM) were designed. The primer and probe concentrations and cycling conditions were extensively optimized and the optimized reaction mixture contained 5 µL of 4X TaqMan Fast Virus 1- Step Master Mix (Applied Biosystems™); 10μM of forward and reverse primers, and 10 μM of probe; 5 µL of extracted DNA and DNase & RNase-free water in a 20 µL reaction. The optimal thermal profile for the VNUA-54 real-time PCR assay is 50°C for 5 minutes; 95°C for 20 seconds; followed by 40 cycles of amplification (3 seconds at 95°C and 30 seconds at 58oC). All real-time PCR reactions were performed on a LightCycleTM 96 (Rocher, Switzerland) and data was analyzed by the manufacturer’s software. For comparison, an ASF real-time PCR developed by Tignon et al., was used as described (Tignon et al., 2011).