Quantifying Toxoplasma gondii in Genomic Samples

Genomic DNA was extracted from the collected samples using the DNeasy^® Blood and Tissue Kit (Qiagen) according to the manufacturer’s instructions. Parasite quantification was carried out by qPCR using primer pairs for the 529-bp repetitive element of T. gondii [37 (link)]. DNA samples were adjusted to 20 ng/μL, and reactions were performed in a final volume of 25 μL using GoTaq^® qPCR Master Mix (Promega, Alcobendas, Madrid, Spain), 10 pmol of each primer and 100 ng of DNA in an Applied Biosystems 7500 FAST Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Amplification was performed by a standard protocol (10 min at 95 °C, 40 cycles at 95 °C for 15 s, and 60 °C for 1 min). The number of T. gondii tachyzoites was calculated by interpolating the average Ct values on a standard curve equivalent to 1 × 10⁵ − 1 × 10⁻¹ tachyzoites generated by tenfold serial dilutions of parasite DNA in a solution of ovine genomic DNA at 20 ng/μL. Parasite proliferation was expressed as the parasite number/ng of DNA. Standard curves for T. gondii showed an average slope always close to −3.3 and an R² > 0.98.