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).