Figure 2

Selection of the primer set for amplification of the BLV LTR region. (A) Touch-down PCR was performed using 72 primer sets with 49 primers designed by the CoCoMo program as shown in Table 1. PCR products were detected by electrophoresis on a 3% agarose gel. Lanes 1-72, 1-72 primer set ID; +, results positive for PCR product; -, negative results for same. *, designates PCR products that were detected but for which the amplicon sizes differed from the predicted size. (B) Summary of results shown in (A). Primer set IDs are arranged according to the degeneracy of the primer set and size of the PCR products. (C) The 4 representative melting curves with 16 primer sets of: BLV-infected BLSC-KU-17 cells (a), BLV-free normal cattle cells (b), and reagent-only as negative control (c). The specificity of the 16 selected primer sets was checked by melting curve analysis. Each PCR amplification was followed by gradual product melting at up to 95°C. (D) The optimization of PCR amplification with primer set ID 15 (CoCoMo 6 and 81). The melting curve of PCR products from BLV-infected BLSC-KU-17 cells (a), the BLV-free normal cattle Ns118 (b), and reagent-only as negative control (c).