Introduction
The expansion of Free Trade Agreements (FTAs) has significantly increased seafood imports, raising concerns about mislabeling of country of origin and consumer safety. Sea cucumbers are imported in substantial quantities, exceeding 1,000 tons annually, in various forms such as dried, frozen, salted, or preserved in brine. The emergence of incidents involving origin fraud underscores the necessity for accurate and efficient molecular identification tools. This study aimed to develop and validate multiplex PCR and peptide nucleic acid (PNA)-based real-time PCR assays for species-level identification of imported and distributed sea cucumbers in the domestic market.
Materials and methods
A total of 32 sea cucumber samples were collected from various retail sources, including domestic supermarkets, traditional markets, and online shopping platforms. Species identification was carried out using small subunit ribosomal RNA (SSU rRNA) gene sequencing, which identified four species: Cucumaria miniata (Red sea cucumber), Stichopus horrens (Ruby cucumber), Apostichopus japonicus (Japanese sea cucumber), and Holothuria spinifera (Brown sea cucumber). Based on these results, four species-specific primer pairs were designed for multiplex PCR, along with four corresponding PNA probes for real-time PCR analysis.
Results and discussion
The developed multiplex PCR assay demonstrated high specificity, with each primer set amplifying only the target species without cross-reactivity. The detection sensitivity ranged from 1 × 10³ to 1 × 10⁴ copies/µL. Similarly, the PNA-based real-time PCR assay showed specific amplification with distinct Tm values for each species, also within the sensitivity range of 1 × 10³ to 1 × 10⁴ copies/µL. These findings suggest that both methods are robust and reliable for species identification. The assays developed in this study are expected to provide effective tools for the authentication of sea cucumber species and for preventing country-of-origin mislabeling in the seafood market.