DNA extraction and molecular detection of Besnoitiaspp.
Total DNA was extracted from an aliquot of ~200 mg of
fresh faecal material using the QIAamp® DNA Stool Mini
Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions.
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.