Primer Effectiveness Test for DNA Authentication of Skipjack Tuna (Katsuwonus pelamis) Using Multiplex PCR

Abstract

Species substitution in processed fishery products remains a major concern for food authenticity and consumer protection, particularly in tuna commodities where mislabeling is frequently reported. Reliable molecular tools are therefore needed to ensure accurate species identification and support food fraud prevention. This study aimed to develop a novel species-specific primer for the molecular authentication of skipjack tuna (Katsuwonus pelamis) and assess its application in a multiplex PCR assay. Three primer candidates targeting mitochondrial DNA regions (12S rRNA and NADH4) were designed and evaluated through in silico BLAST analysis and singleplex PCR. Among the three primer candidates, only one NADH4-based primer pair generated the expected 475 bp amplicon and showed specific amplification of K. pelamis without cross-amplification in non-target species. The selected primer was combined with published primers for Thunnus albacares and Thunnus alalunga in a multiplex PCR assay. Reliable amplification was obtained for K. pelamis and T. albacares, while amplification of T. alalunga was less consistent under multiplex conditions, suggesting that further optimization may improve assay performance. Sensitivity testing demonstrated successful DNA detection at concentrations as low as 0.1 ng/μL. Application of the multiplex PCR assay to processed fish meatballs successfully detected K. pelamis DNA in a complex food matrix. These findings indicate that the developed NADH4-based primer is a reliable molecular marker for K. pelamis authentication. Furthermore, the multiplex PCR approach shows strong potential as a sensitive tool for food authenticity verification and food fraud detection in processed fishery products.