The presence of ITS-1 DNA in the blood means that parasitemia is present and that the latent infection has been successfully activated. DNA was extracted from the peripheral blood of the mice using a DNA extraction kit (AG, Hunan, China) according to the manufacturer’s protocol. PCR amplification of the ITS-1 gene was performed using the primers ITS-1 5′-GATTTGCATTCAAGAAGCGTGATAGTAT-3′ and ITS-1 reverse 5′-AGTTTAGGAAGCAATCTGAAAGCACATC-3′ [19 (link),20 (link)]. The PCR mix consisted of 12.5 μL of PCR Master Mix 1× (Promega, WI, USA), 1 μL of each primer at 1 μM, 2.5 μL of 1 × PCR buffer, and 6 μL of DNA sample in a final volume of 25 μL. The reactions were performed in a thermal cycler (Biometra, Gottingen, Germany) with an initial denaturation step of 95 °C for 3 min. This step was followed by 35 cycles, with 1 cycle consisting of 30 s at 55 °C under the annealing temperature and 30 s at 72 °C. DNA loading buffer (AG, Hunan, China) was added to the amplified PCR product, which was then separated via electrophoresis on a 1% agarose gel containing ethidium bromide and recorded using a digital gel documentation system (BioRad, CA, USA). The positive control was the T. gondii Chinese 1 genotype wh6 strain.
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