Sarcocystis DNA Isolation and Sequencing

The DNA isolation from sarcocysts was conducted using the GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania) according to the manufacturer’s recommendations. The partial 28S rRNA was amplified using KL-P1F/KL-P1R primer pairs (Table 1) [35 (link)]. Each PCR reaction was carried out in a 25 µL mixture containing 12.5 µL of DreamTaq PCR Master Mix (Thermo Fisher Scientific Baltics, Vilnius, Lithuania), 4 µL of DNA template, 0.5 µM of both forward and reverse primers and nuclease-free water. The PCR was initiated with the initial hot start at 95 °C for 5 min followed by 35 cycles of 94 °C for 45 s, annealing at 52 °C for 60 s and 72 °C for 80 s, and a final extension at 72 °C for 7 min. The visualisation, purification, and sequencing of PCR products followed the previously described protocol [36 (link)]. In order to detect essentially similar DNA sequences, and evaluate the interspecific and intraspecific genetic variability of detected Sarcocystis parasites, the 28S rRNA sequences generated in this study were compared with those of various Sarcocystis spp. using the nucleotide BLAST program (http://blast.ncbi.nlm.nih.gov/, accessed on 10 March 2024).