Ultrapure agarose product

PCR amplification was used to determine the presence of genomic DNA from the genus Eimeria [11 (link)]. In a 25 μL reaction mixture, each reaction comprised 100 ng of templates, 10 μM each of forward and reverse primers, 0.5 μL of MyTaq HS DNA polymerase, and 5 μL of 5X MyTaq Reaction Buffer (Bioline, UK). An initial denaturing phase at 95°C for 3 min was followed by 35 cycles at 95°C for 1 min, 55°C for 1 min, 72°C for 2 min, and a final extension at 72°C for 5 min. At 100 V, electrophoresis with 1.5% agarose (UltraPure™ Agarose product, Invitrogen, US) and SYBR Safe DNA Gel Stain was performed (Thermo Fisher Scientific, Finland). An ultraviolet transilluminator was used to visualize the bands.

The nested PCR procedure using ITS-1 primers was standardized for the detection of bovine Eimeria spp. [11 (link)]. Species-specific primers targeting the ITS-1 region were used to amplify the distinct Eimeria spp. [11 (link)]. The product of the primary PCR (first, genomic) (1 L in 25 μL of reaction mixture) was used as a template for the nested PCR (second, species specific) with six species-specific primers (E. bovis, E. zuernii, E. alabamensis, E. auburnensis, E. ellipsoidalis, and E. cylindrica) in individual tubes using the same amplification conditions described above (first PCR). Negative, no-template controls were supplied with each experiment, and distilled water was used in place of the template. Gel electrophoresis in 1.5% agarose (UltraPure™ Agarose product, Invitrogen) was used to verify the amplification of certain nested multiplex PCR products.