Agilent pcr system

The DNA of ASFV were extracted from infected pig-organ samples using PureLink Viral RNA/DNA Mini Kit (Invitrogen, Carlsbad, CA, USA) as given by the manufacturer's instructions. Conventional PCR were performed to amplify the partial gene CD2v (CP204L) gene from Vietnam ASFV strains using the specifi c primer set CD2-2F and 2R, to amplify a 816-bp fragment of EP402R gene coding for the cytoplasmic portion of CD2-like protein as described previously [5, 11] . In the second conventional PCR, we designed a set of primers to amplify of short fragment CDS of CD2v gene, named CD2v-F1 (5'-ATTTTTCCTCATAATGATGTATTTGAT-3', binding site 73,644 -73,670) and CD2v-R1 (5'-TGATATTTGGGGGAGTAGCAGG-3', binding site 73,729-73,751), to amplify a 107-bp fragment of EP402R gene. Primer binding sites were based on the comparison of the genome of the ASFV from China (MK333180.1), Vietnam (MN711757-86), Georgia (FR682468), Russia (KM609342) and Belgium (MK543947). PCR was carried out in Agilent PCR System (Agilent, Santa Clara, CA, USA) using Taq polymerase (Thermo Scientifi c, Waltham, MA USA), according to the manufacturer's instructions. Thermal conditions for performing PCR are as follows: an initial incubation at 95°C for 10 min; 40 cycles of denaturation at 95°C for 15 s, annealing at 58°C for 30 s, and extension at 72°C for 30 s; and fi nal incubation 72°C for 7 min.