2.5. Restriction Fragment Length Polymorphism (RFLP) Analysis

After incubation at 37°C for 3 hours and heat inactivation of RsaI at 65°C for 15 minutes, the digested DNA samples were analyzed by gel electrophoresis, 8μl of each product, and were electrophoresed on 1.5% agarose gel in TBE buffer at 100 V for 60 minutes and visualized by UV illumination. The size of each band was determined by a 100 bp plus ladder molecular weight marker (Thermo Fisher Scientific, USA). DNA types of Fasciola spp. were distinguished according to fragment patterns, three bands of 360, 100, and 60 bp fragments in F. hepatica, while F. gigantica had a profile of 360, 170, and 60 bp in size [11].