Abstract
BackgroundAnisakis 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.MethodsIn 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.ResultsIn 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.ConclusionsThis 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.
Highlights
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 analysis showed that the larvae belong to the species A. simplex s. s. (Supplemental Table 1)
The real-time PCR method used for quantifying gene expression levels requires suitable reference genes as internal controls
Summary
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. 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 to investigate the gene expression at different developmental stages of this parasite. The investigation of the expression of genes at different developmental stages of this parasite seems to be important. It will affect the better understanding of the molecular biology of these parasitic nematodes and will allow finding ways to overcome the disease caused by them. A real-time polymerase chain reaction (real-time PCR) is a valuable method to quantify gene expression [8] at different developmental stages [9, 10]
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