Department of Infectious Diseases and Pathobiology, Institute of Parasitology, University of Bern, Bern, Switzerland.
Graduate School for Cellular and Biomedical Sciences, University of Bern, Bern, Switzerland.
PLoS One. 2022 Sep 16;17(9):e0271011. doi: 10.1371/journal.pone.0271011. eCollection 2022.
Herein, we developed a single and a duplex TaqMan quantitative PCR (qPCR) for absolute quantification of copy numbers of integrated dihydrofolate reductase-thymidylate synthase (mdhfr-ts) drug selectable marker for pyrimethamine resistance in Toxoplasma gondii knockouts (KOs). The single TaqMan qPCR amplifies a 174 bp DNA fragment of the inserted mdhfr-ts and of the wild-type (WT) dhfr-ts (wtdhfr-ts) which is present as single copy gene in Toxoplasma and encodes a sensitive enzyme to pyrimethamine. Thus, the copy number of the dhfr-ts fragment in a given DNA quantity from KO parasites with a single site-specific integration should be twice the number of dhfr-ts copies recorded in the same DNA quantity from WT parasites. The duplex TaqMan qPCR allows simultaneous amplification of the 174 bp dhfr-ts fragment and the T. gondii 529-bp repeat element. Accordingly, for a WT DNA sample, the determined number of tachyzoites given by dhfr-ts amplification is equal to the number of tachyzoites determined by amplification of the Toxoplasma 529-bp, resulting thus in a ratio of 1. However, for a KO clone having a single site-specific integration of mdhfr-ts, the calculated ratio is 2. We then applied both approaches to test T. gondii RH mutants in which the major surface antigen (SAG1) was disrupted through insertion of mdhfr-ts using CRISPR-Cas9. Results from both assays were in correlation showing a high accuracy in detecting KOs with multiple integrated mdhfr-ts. Southern blot analyses using BsaBI and DraIII confirmed qPCRs results. Both TaqMan qPCRs are needed for reliable diagnostic of T. gondii KOs following CRISPR-Cas9-mediated mutagenesis, particularly with respect to off-target effects resulting from multiple insertions of mdhfr-ts. The principle of the duplex TaqMan qPCR is applicable for other selectable markers in Toxoplasma. TaqMan qPCR tools may contribute to more frequent use of WT Toxoplasma strains during functional genomics.
本文开发了一种单重和双重 TaqMan 定量 PCR（qPCR），用于绝对定量整合二氢叶酸还原酶-胸苷酸合酶（mdhfr-ts）药物选择标记物的拷贝数，该标记物对弓形虫敲除体（KOs）中的嘧啶耐药。单重 TaqMan qPCR 扩增插入的 mdhfr-ts 和野生型（WT）二氢叶酸还原酶（wtdhfr-ts）的 174 bp DNA 片段，wtdhfr-ts 作为单个拷贝基因存在于弓形虫中，并编码对嘧啶耐药的敏感酶。因此，在具有单个定点整合的 KO 寄生虫中，给定 DNA 量中的 dhfr-ts 片段的拷贝数应该是 WT 寄生虫中相同 DNA 量记录的 dhfr-ts 拷贝数的两倍。双重 TaqMan qPCR 允许同时扩增 174 bp dhfr-ts 片段和弓形虫 529-bp 重复元件。因此，对于 WT DNA 样本，dhfr-ts 扩增确定的速殖子数量等于扩增弓形虫 529-bp 确定的速殖子数量，因此比值为 1。然而，对于具有单个定点整合的 mdhfr-ts 的 KO 克隆，计算出的比值为 2。然后，我们应用这两种方法来测试通过插入 mdhfr-ts 破坏主要表面抗原（SAG1）的 RH 突变体弓形虫。两种检测方法的结果均相关，显示出在检测具有多个整合的 mdhfr-ts 的 KO 方面具有很高的准确性。使用 BsaBI 和 DraIII 的 Southern blot 分析证实了 qPCR 结果。对于 CRISPR-Cas9 介导的诱变后，T. gondii KOs 的可靠诊断需要使用两种 TaqMan qPCR，特别是要考虑到由于多个插入 mdhfr-ts 而导致的脱靶效应。双重 TaqMan qPCR 的原理适用于弓形虫中的其他选择标记物。TaqMan qPCR 工具可能有助于在功能基因组学中更频繁地使用 WT 弓形虫株。
