A Multiplex PCR Assay Combined with Capillary Electrophoresis for the Simultaneous Identification of Atlantic Cod, Pacific Cod, Blue Whiting, Haddock, and Alaska Pollock

Abstract

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.

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

“…Compared to previous study [35], the primers developed in this study could detect bigeye, skipjack, albacore, and yellowfin tunas' DNA even in low concentrations. Conventional PCR has been reported to be less specific and sensitive than real-time PCR [35,36]. However, the assay conducted in this study based on conventional PCR had higher detection sensitivity than reported by previous studies.…”

“…Capillary electrophoresis has been reported to provide higher resolution than agarose-based electrophoresis when distinguishing DNA amplicons [36]. Then, the multiplex PCR method coupled with capillary electrophoresis was conducted to detect the five species of tunas [36]. The sensitivity of the developed multiplex PCR detected by capillary and agarose-based electrophoreses was 1 pg (Figures 4 and 5).…”

“…Additionally, this assay displays a sensitivity of 1 pg, which provides accuracy and sensitivity in the detection and differentiation of the target species from the five tuna The sensitivity of multiplex PCR was confirmed by gel (Figure 4) and capillary electrophoreses (Figure 5) using diluted DNA (10 ng to 0.01 pg). Capillary electrophoresis has been reported to provide higher resolution than agarose-based electrophoresis when distinguishing DNA amplicons [36]. Then, the multiplex PCR method coupled with capillary electrophoresis was conducted to detect the five species of tunas [36].…”

Capillary electromigration methods for food analysis and Foodomics: Advances and applications in the period March 2021 to March 2023

“…The putative candidate genes identified in this work could be useful for enhancing the shelf life of dessert-type cantaloupe melons. CE methodologies applied to DNA analysis reported in the last 2 years have been aimed at assessing food safety [57][58][59], detecting genetically modified organisms (GMOs) [60,61], evaluating food authenticity [62,63], and detecting food fraud [64,65].…”

Rapid On-Site Identification for Three Arcidae Species (Anadara kagoshimensis, Tegillarca granosa, and Anadara broughtonii) Using Ultrafast PCR Combined with Direct DNA Extraction

“…PCR-based techniques, such as DNA barcoding, PCR restriction fragment length polymorphism, forensically informative nucleotide sequencing, and recombinase polymerase amplification, have been developed to detect different species [ 11 , 12 , 13 , 14 ]. These techniques have been used to identify commercially important seafood species, such as shrimps, cods, crabs, and oysters [ 8 , 15 , 16 , 17 ]. Recently, more rapid and convenient technologies have been developed to confirm the presence of target species in processed foods [ 18 , 19 ].…”