Development and validation of multiplex PCR assay for differentiating tunas and billfishes

Kim, Na-Ye-Seul; Park, Eun-Ji; Lee, Seo-Hyun; Mun, Kwang-Ho; Yang, Ji-Young; Kim, Jung-Beom

doi:10.1007/s10068-021-00893-0

Cited by 6 publications

(3 citation statements)

References 28 publications

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“…The sensitivity of the developed multiplex PCR detected by capillary and agarose-based electrophoreses was 1 pg (Figures 4 and 5). Similar studies have reported a sensitivity of 5 ng for tuna and billfish and 1 ng for blowfish and freshwater fish species [26,37,38]. Our assay demonstrates better sensitivity than those in previously reported studies.…”

Section: Specificity and Sensitivity Of Multiplex Pcrsupporting

confidence: 74%

“…In addition, mitochondria remain after processing, which avoids DNA loss, and since mitochondrial DNA is more abundant than nuclear DNA, it is easier to detect [20,21]. Real-time polymerase chain reaction (PCR) [4,5,[22][23][24], multiple PCR [19,21,25,26], loop-mediated isothermal amplification (LAMP) [6], PCR restriction fragment length polymorphism (PCR-RFLP) [7,27], and others have been reported as viable DNA-based PCR techniques for tuna species identification. Real-time PCR assays require an expensive machine and have the disadvantage that it must be done by skilled experimenters.…”

Section: Introductionmentioning

confidence: 99%

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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)

Lee

Suh

Lee

et al. 2022

Foods

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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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Section: Specificity and Sensitivity Of Multiplex Pcrsupporting

confidence: 74%

Section: Introductionmentioning

confidence: 99%

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

Lee

Suh

Lee

et al. 2022

Foods

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show abstract

“…This result indicated that the designed primers did not inhibit each other in a single reaction and had high specificity, resulting in the sensitivity that corresponded to that of the simplex PCR. Compared to the LOD of similar studies of fish products (5 ng for tunas and billfishes, 1 ng for freshwater fish species, and 1 ng for puffer fish), the LOD of our multiplex PCR assay (1 pg) is very sensitive and can detect very small amounts of cod DNA [21][22][23]. These results suggest that this PCR assay can be applied to on-site detection with low DNA extraction efficiency or to fragmented DNA samples such as processed food.…”

Section: Specificity and Sensitivity Of Multiplex Pcrmentioning

confidence: 77%

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

Lee

Kim

2021

Foods

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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.

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Development of a multiplex PCR assay for the simultaneous detection of big blue octopus (Octopus cyanea), giant Pacific octopus (Enteroctopus dofleini), and common octopus (Octopus vulgaris)

Lee

Kim

2022

Food Sci Biotechnol

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Development and validation of multiplex PCR assay for differentiating tunas and billfishes

Cited by 6 publications

References 28 publications

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

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

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

Development of a multiplex PCR assay for the simultaneous detection of big blue octopus (Octopus cyanea), giant Pacific octopus (Enteroctopus dofleini), and common octopus (Octopus vulgaris)

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