Development of a MGB probe based qPCR protocol for detecting Pacific cod Gadus macrocephalus in eDNA samples

Abstract

The Jinhaeman Bay, located in the South Sea of ​​Korea, is a well-known large-scale Pacific cod (Gadus macrocephalus) spawning ground where it migrates in the winter. We applied an eDNA approach to determine when G. macrocephalus migrates for spawning in the Jinhaeman Bay. We developed novel species-specific primers and minor groove binding (MGB) probe sets for quantitative PCR (qPCR) assays targeting the cytochrome b gene sequences. The cross-species test revealed that the assay not only detects the DNA of the target species but also detects low concentrations with high specificity. Moreover, the assay was also applied to water collected from the Jinhaeman Bay during the G. macrocephalus spawning season, and successful DNA amplification of the target species was confirmed. The assay was tested in silico, in vitro, and in vivo. Through these evaluations, the species specificity of the eDNA method for G. macrocephalus was confirmed, and this method was demonstrated to be useful for rapid assessment of the presence of this species. In particular, this method has been shown to be useful to determine when this species starts entering the spawning grounds and to characterize what its distribution is within the spawning grounds. © 2023, The Author(s), under exclusive licence to Springer Nature B.V.