ITS1 Amplification of Ancylostoma Parasites

The ITS1 sequences of A. ceylanicum and A. caninum were amplified using a forward primer AF (5′-CTTTGTCGGGAAGGTTGG-3′) and a reverse primer AR (5′-TTCACCACTCTAAGCGTCT-3′) designed by Liu et al. [15 (link)]. The predictive amplification fragment was 404 bp. Polymerase chain reactions were performed in 25 μL, including 12.5 μL of Premix Ex-Taq polymerase (TaKaRa, Dalian, China), 9.5 μL of ddH₂O, 0.5 μL of each primer AF/AR (50 μmol/L), and 2 μL of DNA sample. PCR cycling parameters were as follows: 1 cycle at 94°C for 5 min and then 35 cycles at 94°C for 30 sec, at 61.5°C for 30 sec, and at 72°C for 45 sec, followed by 1 cycle at 72°C for 7 min. The PCR products were analysed by gel electrophoresis in 1.5% agarose gels, stained with 0.2 mg/ml ethidium bromide, and visualized on a UV transilluminator.