Real-time PCR and molecular genotyping of ASFV
DNA was extracted directly from the blood using a nucleic acid
extraction kit (DNeasy Blood & Tissue kit, Qiagen, Hilden, Germany)
following the manufacturer’s procedures. The TaqMan® probe and primers
described previously were used for the detection of 250 bp of 3’-end of
the p72 gene for qPCR (King et al, 2003) for initial diagnosis. Then,
478 bp fragments corresponding to the C-terminal end of the p72 gene
were amplified for genomic sequencing using primers as described
previously (Bastos et al, 2003). The PCR amplicons were purified using a
QIAquick PCR Purification kit (Qiagen, Hilden, Germany). The sequence
cycles were performed with the Genome Lab DTCS-Quick Start Kit (Beckman
Coulter, California, USA) and analyzed by the CEQ-8000 Genetic Analysis
System (Beckman Coulter, California, USA). The sequence data (456 bp) in
this study and thirty partial/complete B646L gene sequences encode for
major ASFV capsid p72 protein as representative from each genotype as
described previously (9 ) were retrieved from the gene bank
aligned and edited to 399 nucleotides for uniformity using the BioEdit
Sequence Alignment Editor (Hall, 1999). The phylogenetic analysis was
conducted by the neighbor-joining approach with 1,000 bootstrap
replications using MEGA X software (Saitou & Mei, 1987; Felsenstein,
1985; Kimura, 1980; Kumar et al, 2018).