Rapid Detection of Epinephelus Species Substitution in the Greek Market Using High-Resolution Melting Analysis

<b>Background/Objectives</b>: Fish are vital in the Mediterranean diet, offering protein, nutrients, and ω-3 fatty acids. Greek consumers favor wild-caught, high-value fish like the dusky grouper (<i>Epinephelus marginatus</i>) classified as "vulnerable" and the white grouper (<i>Epinephelus aeneus</i>) classified as "near threatened" species, according to the IUCN Red List. Due to their premium prices and complex supply chains, these species are susceptible to fraud, especially through mislabeling. This practice not only deceives consumers but also poses health risks and encourages illegal fishing. DNA-based methods have shown effectiveness in accurately identifying species, even in processed samples. The aim of this study is to apply high-resolution melting analysis (HRM) as a rapid, effective method for monitoring the appropriate labeling of the two <i>Epinephelus</i> species in the Greek market. <b>Methods</b>: In this study, fresh fish from Greek catches as well as cooked, frozen, and filleted samples collected from the Greek market were identified using DNA barcoding. HRM analysis based on single nucleotide polymorphisms (SNPs) was used to differentiate between locally sourced <i>E. marginatus</i> and <i>E. aeneus</i> from their imported counterparts or from other species available in the Greek market that could be used in substitution incidents. <b>Results:</b> Using HRM analysis, cases of species mislabeling were identified and were also confirmed using sequencing. <b>Conclusions:</b> HRM analysis proved to be an accurate and cost-effective method for rapidly processing a large number of samples; therefore, it could serve as a valuable tool in extensive market controls as well as for bio-diversity conservation monitoring.