Background

Anisakis simplex s. s. is a parasitic nematode with a complex life cycle in which humans can become accidental hosts by consuming raw or not fully cooked fish containing L3 larvae. The growing popularity of raw fish dishes has contributed to an increase in the incidence of anisakiasis, which has spurred scientific efforts to develop new methods for diagnosing and treating the disease and also to investigate the gene expression at different developmental stages of this parasite. The identification of reference genes suitable for the normalization of RT-qPCR data has not been studied with respect to A. simplex s. s.

Methods

In the present study, eight candidate reference genes were analyzed in A. simplex s. s. at two different developmental stages: L3 and L4. The expression stability of these genes was assessed by geNorm and NormFinder softwares.

Results

In general, our results identified translation elongation factor 1α (ef-1α) and peptidyl-prolyl isomerase 12 (ppi12) as the most stable genes in L3 and L4 developmental stages of A. simplex s. s. Validation of the selected reference genes was performed by profiling the expression of the nuclear hormone receptor gene (nhr 48) in different developmental stages.

Conclusions

This first analysis selecting suitable reference genes for RT-qPCR in A. simplex s. s. will facilitate future functional analyses and deep mining of genetic resources in this parasitic nematode.

中文翻译：

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选择可靠的参考基因用于不同发育阶段异尖线虫 Sensu Stricto 基因表达的 RT-qPCR 分析。

背景

Anisakis simplex ss 是一种具有复杂生命周期的寄生线虫，人类可以通过食用含有 L3 幼虫的生鱼或未完全煮熟的鱼而成为意外宿主。生鱼菜肴的日益流行导致异尖线虫病的发病率增加，这促使科学努力开发诊断和治疗疾病的新方法，并研究这种寄生虫不同发育阶段的基因表达。尚未就A. simplex ss研究适合 RT-qPCR 数据标准化的参考基因的鉴定

方法

在本研究中，在A. simplex ss 中在两个不同的发育阶段分析了八个候选参考基因：L3 和 L4。这些基因的表达稳定性通过geNorm和NormFinder软件进行评估。

结果

一般而言，我们的结果确定翻译延伸因子 1 α ( ef-1α ) 和肽酰脯氨酰异构酶 12 ( ppi12)是A. simplex ss L3 和 L4 发育阶段中最稳定的基因。所选参考基因的验证由分析核激素受体基因 ( nhr 48 ) 在不同发育阶段的表达。

结论

选择合适的参考基因用于单纯放线虫 ss 中的RT-qPCR 的首次分析将有助于未来对该寄生线虫的遗传资源进行功能分析和深度挖掘。