Institute of Parasitology, BFS, Justus Liebig University, Giessen, Germany.
Institute for Parasitology, Centre for Infection Medicine, University of Veterinary Medicine Hanover, Hanover, Germany.
Sci Rep. 2019 Nov 1;9(1):15867. doi: 10.1038/s41598-019-52416-x.
The liver fluke Fasciola hepatica causes fasciolosis, a foodborne zoonosis affecting humans and livestock worldwide. A reliable quantification of gene expression in all parasite life stages relevant for targeting by anthelmintics in the mammalian host is fundamental. The aim of this study was to define a set of stably expressed reference genes for qRT-PCR in Fasciola studies. We determined the expression stabilities of eight candidate reference genes by the algorithms NormFinder, geNorm, BestKeeper, and comparative ΔCT method. The most stably expressed reference genes for the comparison of intra-mammalian life stages were glutamyl-prolyl-tRNA synthetase (Fheprs) and tubulin-specific chaperone D (Fhtbcd). The two best reference genes for analysis of in vitro-cultured juveniles were Fhtbcd and proteasome subunit beta type-7 (Fhpsmb7). These genes should replace the housekeeping gene gapdh which is used in most Fasciola studies to date, but in fact was differentially expressed in our analysis. Based on the new reference genes, we quantified expression of five kinases (Abl1, Abl2, PKC, Akt1, Plk1) discussed as targets in other parasitic flatworms. Distinct expression patterns throughout development were revealed and point to interesting biological functions. We like to motivate using this set of validated reference genes for future F. hepatica research, such as studies on drug targets or parasite development.
肝片形吸虫 Fasciola hepatica 引起片形吸虫病，这是一种食源性病原体，影响全球的人类和家畜。可靠地定量检测与哺乳动物宿主中驱虫药靶标相关的所有寄生虫生活阶段的基因表达是基础。本研究旨在确定一组在 Fasciola 研究中用于 qRT-PCR 的稳定表达参考基因。我们通过 NormFinder、geNorm、BestKeeper 和比较 ΔCT 方法确定了 8 个候选参考基因的表达稳定性。用于比较哺乳动物体内生活阶段的最稳定表达的参考基因是谷氨酰-脯氨酰-tRNA 合成酶 (Fheprs) 和微管蛋白特异性伴侣 D (Fhtbcd)。用于分析体外培养的幼虫的两个最佳参考基因是 Fhtbcd 和蛋白酶体亚基β类型-7 (Fhpsmb7)。这些基因应该取代迄今为止在大多数 Fasciola 研究中使用的管家基因 gapdh，但实际上在我们的分析中差异表达。基于新的参考基因，我们定量了五个激酶 (Abl1、Abl2、PKC、Akt1、Plk1) 的表达，这些激酶在其他寄生扁虫中被认为是靶标。在整个发育过程中揭示了不同的表达模式，并指向有趣的生物学功能。我们希望在未来的肝片形吸虫研究中使用这套经过验证的参考基因，例如药物靶标或寄生虫发育的研究。
