Quantitative Analysis of Toxoplasma Virulence Genes

Total RNA was extracted from 1 × 10⁷ tachyzoites of the Nc-1 wild-type strain and three ΔNcROP5 clones with TRIzol reagent (Invitrogen, USA). cDNA was synthesized using the EasyScript First-Strand cDNA Synthesis SuperMix kit (TransGen, China). Specific primers were designed using Primer Premier 5.0 (Hui et al., 2014 (link)), including primers for rhoptry necks (RON2 and RON4), rhoptrys (ROP4, ROP5, ROP7, ROP16, and ROP17), dense granules (GRA2, GRA6, and GRA7) and the endogenous reference gene NcActin (Supplementary Table S1). The specificity of these primers was evaluated using conventional quantitative real-time PCR (qRT-PCR). The qRT-PCR was conducted using the ABI Prism 7500 System (Biosystems Inc., USA) with SYBR Green II (Takara Biotechnology, Dalian, Co., Ltd) following manufacturer’s instructions. The resulting RNA concentrations were normalized using Ncactin (Wang et al., 2017 (link)), and the relative expression levels of the target genes were analyzed using the ABI Prism 7500 software v2.0.5 (Biosystems Inc., USA). The RT-PCR conditions were as follows: 94°C for 5 s, 40 cycles of 94°C for 5 s and 60°C for 30 s. The relative expression of genes was calculated using the 2^-ΔΔCt method and standard deviation was calculated from three replicates (Cárdenas-Mondragón et al., 2016 (link); Gagnaire et al., 2016 (link)).