A novel multiplex PCR method for the identification of red snapper (Lutjanus purpureus) and detection of multi-species fraud

Abstract The red snapper ( Lutjanus purpureus ) is a highly valued commercial fish species subject to intense global trade and high rates of economic fraud through species substitution. Traditional DNA barcode approaches, while accurate, are costly and time-consuming for routine regulatory enforcement and industrial food control. To address this methodological limitation, we developed and validated a novel, low-cost forensic multiplex PCR screening assay based on the mitochondrial Cytochrome B (Cytb) gene. This high-throughput method simultaneously differentiates four Lutjanidae species from the Western Atlantic coast by generating species-specific diagnostic amplicon sizes: L. purpureus (420 bp), Rhomboplites aurorubens (237 bp), Lutjanus buccanella (237 and 170 bp), and Lutjanus vivanus (420 and 237 bp), alongside a universal internal control (~ 650 bp). To demonstrate the method’s practical applicability in food inspection, the optimized protocol was applied to authenticate commercial fillets labeled as “snapper” (“pargo” in Brazil). The assay, confirmed by DNA sequencing, successfully detected recurrent multi-species fraud, revealing that R. aurorubens remains the primary economic substitute (occurring in 27.9% of the samples), while samples designated for international export showed full authenticity. This novel multiplex PCR assay provides a robust, rapid, and highly sensitive analytical tool that can be easily integrated into routine food laboratories to enforce seafood traceability, combat commercial fraud, and support regulatory compliance.