Pcr master mix 1

DNA extraction from the retina of each mouse was performed using a DNA Extraction Kit (TaKaRa, Japan) in accordance with the manufacturer’s protocol. PCR amplification of the ITS-1 gene [25 (link)] was performed by using the primers ITS-1 5′-GATTTGCATTCAAGAAGCGTGATAGTAT-3′ and ITS-1 reverse 5′-AGTTTAGGAAGCAATCTGAAAGCACATC-3′.The PCR mix consisted of 12.5 μl of PCR Master Mix 1× (Promega, USA), 1 μl of each primer at 1 μM, 2.5 μl of 1× PCR buffer and 8 μl of DNA sample in a final volume of 25 μl. The first step of amplification was 3 min of denaturation at 95°C. This step was followed by 35 cycles, with 1 cycle consisting of 30 s at 55°C at the annealing temperature, and 30 s at 72°C. All PCRs were performed in a thermal cycler (Biometra, Germany). The amplified cDNAs were electrophoretically separated on 1% agarose gels containing ethidium bromide and recorded using a digital gel documentation system (Bio-Rad, USA).

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.