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.

With an increased consumption of seafood products, food fraud with fish resources has been continuously reported. In particular, codfish has been exploited worldwide as a processed product in fresh, frozen, smoked, canned, or ready-to-eat dish forms. However, it is challenging to identify processed fish products after processing because of their similar morphological characteristics. Substitution and mislabeling of codfish among different species are also happening deliberately or unintentionally. Thus, it is necessary to distinguish cod species to prevent fish adulteration and food fraud. In this study, we developed a multiplex PCR for simultaneously identifying five cod species within Gadidae using capillary electrophoresis. Then, their species-specific primer sets were designed by targeting the mitochondrial cytochrome b gene. Subsequently, the amplicon sizes obtained were 237 bp, 204 bp, 164 bp, 138 bp, and 98 bp for Atlantic cod, Pacific cod, blue whiting, haddock, and Alaska pollock, respectively. The specificity of each primer was further tested using 19 fish species, and no cross-reactivity was observed. The limit of detection of this multiplex PCR assay was 1 pg. The developed multiplex PCR assay can be applied to 40 commercial food products successfully. This detection method will be efficient for managing seafood authentication by simultaneously analyzing multiple cod species.

To provide consumers correct information on meat species, specific and sensitive detection methods are needed. Thus, we developed a capillary electrophoresis-based multiplex PCR assay to simultaneously detect red deer (Cervus elaphus), roe deer (Capreolus capreolus), and water deer (Hydropotes inermis). Specific primer sets for these three species were newly designed. Each primer set only amplified target species without any reactivity against non-target species. To identify multiple targets in a single reaction, multiplex PCR was optimized and combined with capillary electrophoresis to increase resolution and accuracy for the detection of multiple targets. The detection levels of this assay were 0.1 pg for red deer and roe deer and 1 pg for water deer. In addition, its applicability was demonstrated using various concentrations of meat DNA mixtures. Consequently, as low as 0.1% of the target species was detectable using the developed method. This capillary electrophoresis-based multiplex PCR assay for simultaneous detection of three types of deer meat could authenticate deer species labeled on products, thus protecting consumers from meat adulteration.