Toxoplasma gondii DNA

Meister, Seraina L., Wenker, Christian, Wyss, Fabia, Zühlke, Irene, In, Veiga, es Berenguer & Basso, Walter U., 2022, Syngamus trachea in free-ranging white stork (Ciconia ciconia) nestlings in Switzerland, International Journal for Parasitology: Parasites and Wildlife 18, pp. 76-81 : 77-78

2.6.2. Toxoplasma gondii DNA detection

A real-time PCR targeting the B1 gene (126 bp) of T. gondii was performed ( Costa et al., 2000) on a LightCycler® Instrument (Roche

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Diagnostics, Basel, Switzerland). One primer set (23-mer, sense: 5 ′ -GGAGGACTGGCAACCTGGTGTCG-3 ′ and 25-mer, antisense: 5 ′ -TTGTTTCACCCGGACCGTTTAGCAG-3 ′) was used at a 20 μM working solution as well as a pair of LightCycler® hybridization probes (5 ′ -ACGGGCGAGTAGCACCTGAGGAGAT-3 ′ and 5 ′ -CGGAAATAGAAAG CCATGAGGCACTCC-3 ′) at a 2.5 μM working solution. The amplification mixture consisted of 0.25 μl of each primer, 1.0 μl of each probe, 1.0 μl of a commercial master mix (LightCycler® DNA Master Hybridization Probes Kit, Roche Diagnostics), 0.8 μl MgCl 2 (25 mM), 4.575 μl H 2 O and 1 μl of template DNA in a final volume of 10 μl. Carry-over contamination was prevented by using 0.125 μl of heat-labile uracyl-DNA-glycosylase (UDG) per tube. The samples were amplified using the following thermal profile: 40 ◦ C for 10’ (to allow the UDG to act), 95 ◦ C for 15’ (for denaturation of the template DNA, inactivation of the UDG enzyme and activation of the FastStart Taq DNA Polymerase), followed by 50 cycles of denaturation at 95 ◦ C for 10 ′′, annealing at 56 ◦ C for 20 ′′ and extension at 72 ◦ C for 20 ′′. A known positive DNA control and a negative control (ddH 2 O) were used for each run.