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).