The presence of SFV DNA was determined by using nested PCR. Five hundred ng purified DNA was combined with a PCR reaction mixture with a final concentration of 10 mmol/L Tris (pH 9.0), 50 mmol/L KCl, 0.1% Triton X-100, 2 mmol/L MgCl 2 , 200 µmol/L each dNTP, 0.15 mg/mL BSA, 1 µm Taq polymerase, and 400 nmol/L of each primer in a total volume of 50 µL. The following primer pairs were used: first round, forward 5′ CAG TGA ATT CCA GAA TCT CTT C 3′, reverse 5′ CAC TTA TCC CAC TAG ATG GTT C 3′; and second round, forward 5′ CCA GAA TCT CTT CAT ACT AAC TA 3′, reverse 5′ GAT GGT TCC CTA AGC AAG GC 3′ (29) . "Touchdown PCR" was used for both rounds with reaction conditions of initial denaturation at 94°C for 2 min, followed by 7 cycles of 94°C for 30 s, 60°C for 30 s, and 72°C for 45 s, with a 2°C decrease in annealing temperature per cycle to 48°C, followed by 33 cycles of 94°C for 30 s, 48°C for 30 s, and 72°C for 45 seconds with a final extension at 72°C for 2 min. Second-round conditions were the same, except 19 cycles were used instead of 33.