PCR Amplification of T. parva p67 Gene

PCR amplification targeting the 900 bp variable region of the p67 encoding gene was performed on 232 T. parva positive DNA samples (Table 1), using the primer pair IL613 (5’-ACAAACACAATCCCAAGTTC-3’) and IL792 (5’-CCTTTACTACGTTGGCG-3’) [22 (link)] and the 2X Phusion™ Flash High-Fidelity PCR Master Mix (ThermoFisher Scientific™, Waltham MA, USA) containing Phusion Flash II DNA polymerase which has proof-reading activity [37 (link)]. At least 50 ng of DNA and 10 pmol of each primer were used in a total reaction volume of 12.5 μl. The amplification conditions were as previously described [22 (link)], with some modifications in accordance with the Phusion Flash High-Fidelity PCR Master Mix conditions. Thus, the initial denaturation was done at 98°C for 10 seconds (s), followed by 30 cycles of denaturation at 98°C for 1 s, annealing at 57°C for 5 s and extension at 72°C for 10 s, then one cycle for the final extension step at 72°C for 1 minute (min). Samples that failed to amplify in the primary reaction were re-amplified in the second PCR using 0.5 μl of the primary PCR product as DNA template, and the same amplification conditions except that the amplification cycles were reduced to 20. PCR products were resolved by gel electrophoresis using 2% agarose stained with ethidium bromide.