Molecular detection of parasites by PCR
To generate a positive control for the PCR analysis, genomic DNA extracted from in vitro cultures of various parasites and kindly provided by collaborators was used: Trypanosoma brucei ,Plasmodium falciparum , Neospora caninum , Sarcocystis gigantea , Toxoplasma gondii , Babesia bovis andTheileria orientalis . The negative control consisted of the PCR mix with nuclease-free water added instead of gDNA. PCR primers targeting a conserved region of the 18S subunit of the ribosomal RNA gene (18S rRNA), or the cytochrome b gene in the case ofPlasmodium , were obtained from the literature for the seven parasite genera selected in this study (Table 1 and references therein).
PCR amplification was performed in a 25 μl reaction mixture containing 1x Green GoTaq Flexi buffer, 2 mM MgCl2, 10 mM dNTPs, 0.2 μM of both forward and reverse primers (Table 1), 0.625 units of GoTaq G2 DNA polymerase (Promega, USA), and 1 μl of total genomic DNA template. The PCR program consisted of an initial denaturation step at 95ºC for 2 minutes, followed by 35 cycles of denaturation at 95ºC for 45 s, annealing at 55–60ºC for 45 s and extension at 72ºC for 45–90 s, with a final extension of 5 min at 72ºC (see Table 1). Amplification occurred in a T100 thermal cycler (BioRad, USA). Amplification products were visualised by gel electrophoresis, using a 2% agarose gel, RedSafe™ (iNtRON Biotechnology, Korea), and a high-resolution imaging system (ChemiDoc™ MP Imaging System, Bio-Rad, USA).