Method for the differentiation of nine species of Sebastes with fluorescence melting curve analysis and dual-labeled probes

Abstract

Substitution of a high-value fish species and their products with a low-value species has recently emerged as a problem in the fishery industry, and the need for development of analysis tools for the identification of different species has been discussed globally. The genus Sebastes is the most species-rich of the family Scorpaenidae, and many of the species of Sebastes are commercially and ecologically important, particularly in the North Pacific. They exhibit high morphological similarity among species, and are difficult to distinguish visually. In this study, we selected nine common Sebastes species (S. pachycephalus, S. fasciatus, S. inermis, S. oblongus, S. owstoni, S. hubbsi, S. schlegeli, S. vulpes, and S. thompsoni) for development of a differentiation assay. Fluorescence melting curve analysis (FMCA) was used to distinguish the nine species of Sebastes with single locked nucleic acids (LNA). Dual-labeled probes were designed from the mitochondrial cytochrome c oxidase subunit I gene to detect each species and hybridize with the target sequence. In addition, we successfully multiplex analyzed three species of Sebastes using the fluorescent dyes FAM, HEX, and Cy5. The LNA-based FMCA method we developed is a powerful tool for the identification of nine different species of Sebastes.