2.5. Polymerase chain reaction (PCR)
For the molecular detection of T. gondii , the real-time PCR was performed in the CFX96 Touch Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, California USA) using targeted a 529 bp repeat element and internal transcribed spacer-1 (ITS-1) region-specificT. gondii primers listed in Table 1. The reaction mixture contained 15 μL of dH2O, 5 μL of 5X Hot Start Master Mix (GMbiolab, Taiwan, China), 2 μL of 10 μmol/L forward and reverse primer, and 3 μL DNA Template. The thermal cycling was performed at 37 cycles (Enzyme activation at 95°C for 5 minutes, denaturing at 95°C for 30 seconds, annealing at 56°C for 30 seconds and extension at 72°C for 1 minute). Melting curves were generated from 50 to 95°C and read every 4°C for 5 seconds.
Table 1. Nucleotide sequences of primers used in this study