Multiplex PCR Assay for Simultaneous Identification of Five Types of Tuna (Katsuwonus pelamis, Thunnus alalonga, T. albacares, T. obesus and T. thynnus)
Abstract
:1. Introduction
2. Materials and Methods
2.1. Samples
2.2. DNA Extraction
2.3. Species-Specific Multiplex Primer Design
2.4. Single and Multiplex PCR
3. Results and Discussion
3.1. Species-Specific Multiplex Primer Design
3.2. Specificity and Sensitivity of Single PCR
3.3. Specificity and Sensitivity of Multiplex PCR
3.4. Application and Validation toward Commercial Food Products
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Target Species | Target Gene | Primer Name | Sequence (5′ → 3′) | Amplicon Size (bp) | Concentration (μM) | Accession No. | Reference |
---|---|---|---|---|---|---|---|
Thunnus obesus | ATP6 | Obe-F | ACT TGC ATT CCC CCT ATG G | 270 | 1.4 | KY400011.1 | This study |
Obe-R | GCT GTT AGG ATT GCC ACA G | ||||||
Katsuwonus pelamis | Cytb | Kat-F | GGT CCT AGC TCT TCT TGC A | 238 | 1.2 | NC_005316.1 | This study |
Kat-R | TGC AAG TGG GAA GAA GAT G | ||||||
Thunnus thynnus | NADH5 | Thy-F | AAC TCT TTA TCG GGT GGG AG | 200 | 0.4 | KF906720.1 | This study |
Thy-R | 1 AGC GGT TAC GAA CAT TTG CTT C | ||||||
Thunnus alalunga | Cytb | Ala-F | GTT TCG TGA TCC TGC TAG TG | 178 | 0.6 | NC_005317.1 | This study |
Ala-R | CCT CCT AGT TTG TTG GAA TAG AT | ||||||
Thunnus albacares | NADH4 | Alba-F | CAT GAT TGC CCA CGG ACT TA | 127 | 1.2 | KM588080.1 | This study |
Alba-R | TGT TGT TAT AAG GGG CAG C |
No | Product Type | Labeled Species | Multiplex PCR Results | ||||
---|---|---|---|---|---|---|---|
Bigeye Tuna | Skipjack Tuna | Atlantic Bluefin Tuna | Albacore Tuna | Yellowfin Tuna | |||
1 | Raw | Bigeye tuna | +++ | ||||
2 | Raw | Bigeye tuna | +++ | ||||
3 | Raw | Bigeye tuna | +++ | ||||
4 | Raw | Bigeye tuna | +++ | ||||
5 | Raw | Bigeye tuna | +++ | ||||
6 | Raw | Bigeye tuna | +++ | ||||
7 | Dried | Skipjack tuna | +++ | +++ | +++ | ||
8 | Canned | Skipjack tuna | +++ | ||||
9 | Canned | Skipjack tuna | +++ | +++ | |||
10 | Canned | Skipjack tuna | +++ | +++ | +++ | ||
11 | Canned | Skipjack tuna | +++ | ||||
12 | Canned | Skipjack tuna | +++ | +++ | |||
13 | Raw | Skipjack tuna | +++ | ||||
14 | Raw | Skipjack tuna | +++ | ||||
15 | Raw | Skipjack tuna | +++ | ||||
16 | Raw | Skipjack tuna | +++ | ||||
17 | Dried | Atlantic bluefin tuna | +++ | ||||
18 | Raw | Atlantic bluefin tuna | +++ | ||||
19 | Raw | Atlantic bluefin tuna | +++ | ||||
20 | Raw | Atlantic bluefin tuna | +++ | ||||
21 | Raw | Atlantic bluefin tuna | +++ | ||||
22 | Canned | Albacore tuna | +++ | ||||
23 | Canned | Yellowfin tuna | +++ | ||||
24 | Raw | Yellowfin tuna | +++ | ||||
25 | Raw | Yellowfin tuna | +++ | ||||
26 | Raw | Yellowfin tuna | +++ | ||||
27 | Raw | Yellowfin tuna | +++ | ||||
28 | Canned | Light Tuna | +++ | +++ | |||
29 | Canned | Light Tuna | +++ | +++ | |||
30 | Canned | Light Tuna | +++ | +++ | +++ | ||
31 | Canned | Light Tuna | +++ | +++ | |||
32 | Canned | Light Tuna | +++ | +++ | +++ |
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Lee, G.-Y.; Suh, S.-M.; Lee, Y.-M.; Kim, H.-Y. Multiplex PCR Assay for Simultaneous Identification of Five Types of Tuna (Katsuwonus pelamis, Thunnus alalonga, T. albacares, T. obesus and T. thynnus). Foods 2022, 11, 280. https://doi.org/10.3390/foods11030280
Lee G-Y, Suh S-M, Lee Y-M, Kim H-Y. Multiplex PCR Assay for Simultaneous Identification of Five Types of Tuna (Katsuwonus pelamis, Thunnus alalonga, T. albacares, T. obesus and T. thynnus). Foods. 2022; 11(3):280. https://doi.org/10.3390/foods11030280
Chicago/Turabian StyleLee, Ga-Young, Seung-Man Suh, Yu-Min Lee, and Hae-Yeong Kim. 2022. "Multiplex PCR Assay for Simultaneous Identification of Five Types of Tuna (Katsuwonus pelamis, Thunnus alalonga, T. albacares, T. obesus and T. thynnus)" Foods 11, no. 3: 280. https://doi.org/10.3390/foods11030280