DNA extraction and molecular detection of Besnoitiaspp.
Genomic DNA was isolated from about 200 mg of faecal material by using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions, except that samples mixed with ASL lysis buffer were incubated for 10 min at 95 °C for improving oocyst wall rupture. Purified DNA samples (200 μl) were stored at –20 °C until downstream PCR-based diagnostic and subtyping analyses were conducted. A water extraction control was routinely included in each sample batch processed for DNA extraction. The products of the DNA extraction process were tested for the specific detection of Besnoitia spp. by ITS-1 rDNA PCR (Cortes et al., 2007). The forward primer ITS1F (50-TGACATTTAATAACAATCAACCCTT-30) and the reverse primer ITS1R (50-GGTTTGTATTAACCAATCCGTGA-30) were added at a concentration of 10 μM, and the rest of reagents were incorporated in the mixture (final volume: 25 μl), as indicated by Frey et al. (2013). The amplified products were visualized after electrophoresis on a 1.5% agarose gel containing 0.1 μl/ml GelRed™ Nucleic Acid Gel Stain (Biotium, Fremont, USA). DNA extraction and PCR were performed in separate laboratories under biosafety level II conditions (BIO II A Cabinet, TELSTAR, Madrid, Spain) to avoid cross contamination. The positive control was DNA extracted from in vitro cultured tachyzoites of B. besnoiti , and PCR grade water was used as the negative control.