Toxoplasma gondii

Gil, Miguel Pardo, Hegglin, Daniel, Briner, Thomas, Ruetten, Maja, Müller, Norbert, Gaston, More, Frey, Caroline F., Deplazes, Peter & Basso, Walter, 2023, High prevalence rates of Toxoplasma gondii in cat-hunted small mammals - Evidence for parasite induced behavioural manipulation in the natural environment?, International Journal for Parasitology: Parasites and Wildlife 20, pp. 108-116 : 110

2.2.4. Genetic characterization of T. gondii

Samples showing DNA-yields with Ct values <34 by T. gondii real-time qPCR were selected for further genotyping. Genetic characterization of T. gondii was performed by multilocus sequence typing (MLST) using nested PCR for 10 genetic markers, including SAG1, SAG2 (5 ′ SAG2, 3 ′ SAG2 and alt. SAG2), SAG3, BTUB, GRA6, c22-8, c29–2, L358, PK1 and Apico, as previously described ( Su et al., 2006, 2010). Subsequently, PCR products were purified (DNA Clean & Concentrator-5, Zymo Research, Irvine, USA) and sequenced bi-directionally with the same primers used in the second step of the nested PCR (Microsynth, Balgach, Switzerland). The obtained marker sequences were aligned and inspected for single nucleotide polymorphisms (SNPs), digested in silico using the NEBcutter V2.0 programme ( Vincze et al., 2003) and the RFLP profiles were analysed as described ( Castro et al., 2020) in order to compare them with the T. gondii genotypes reported in the ToxoDB database (https://toxodb. org/toxo/app).