Quantitative PCR (qPCR) was performed to quantify the expression of uracil phosphoribosyltransferase [(UPRT) (TGGT1_312480)] gene and apicoplast gene (TGGT1_302050) between WT strain and ΔFabZ strain. The 18 s rRNA gene was used as an internal reference for normalization. The qPCR assay was performed as described previously (Wu et al., 2009 (link); Yeh and DeRisi, 2011 (link); Reiff et al., 2012 (link); Bansal et al., 2017 (link)). Genomic DNA was isolated from freshly egressed tachyzoites using Tiangen DNA extraction kit according to the manufacturer’s directions. The apicoplast and UPRT-specific gene sequences were amplified using Max Super-Fidelity DNA Polymerase (Vazyme). The amplification reaction mixture included 10 μl of 2 × SYBR Green pro Taq HS Premix (final concentration 1 ×) (Accurate Biology), 0.4 μl of 10 μM of each primer, 40 ng of the extracted T. gondii DNA, 0.4 μl of ROX Reference Dye, and sterile water to a final volume of 20 μl. qPCR was performed using an initial denaturation at 95°C for 30 s; followed by 40 cycles of amplification at 95°C for 10 s, 56°C for 20 s, and 72°C for 30 s.
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