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There is a need to identify the species of similar types of fish, especially those that are commercially sold. Particularly, the price of tuna varies depending on its type, which is difficult to determine as they are sold in cut or processed forms. This study developed a multiplex polymerase chain reaction (PCR) assay to identify the five most common tuna species: bigeye, skipjack, Atlantic bluefin, albacore, and yellowfin tunas. Newly designed species-specific primer sets for these five tuna species were created. Subsequently, the amplicon sizes obtained were 270, 238, 200, 178, and 127 base pairs for bigeye, skipjack, Atlantic bluefin, albacore, and yellowfin tunas, respectively. Each primer’s specificity was further tested using 15 other fish species, and no cross-reactivity was observed. To identify multiple targets in a single reaction, multiplex PCR was optimized to increase its resolution and accuracy. The detection levels of the multiplex PCR assay were confirmed to be 1 pg for all the five tunas. Additionally, it was successfully applied to 32 types of commercial tuna products. Therefore, this multiplex PCR assay could be an efficient identification method for various tuna species.

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Multiplex PCR Assay for Simultaneous Identification of Five Types of Tuna (Katsuwonus pelamis, Thunnus alalonga, T. albacares, T. obesus and T. thynnus)