Determining the Authenticity of Shark Meat Products by DNA Sequencing
Abstract
:1. Introduction
2. Materials and Methods
2.1. Samples Collection
2.2. Fish Labeling Analysis
2.3. DNA Extraction and Purification
2.4. Oligonucleotide Primers and Reference Genes
2.5. COI and NADH2 PCR Assay
2.6. Detection of Amplified Products
2.7. PCR Cleanup
2.8. Cycle Sequencing Reaction
2.9. Analysis of Sequences
2.10. Assessment Conservation Status
3. Results
3.1. Analysis of the Fish Labels
3.2. Data Analysis
3.3. DNA-Based Species Identification
3.4. Conservation Status of Identified Species
4. Discussion
Supplementary Materials
Author Contributions
Funding
Conflicts of Interest
References
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Target Gene | Primer Sequences (5′–3′) | Amplicon Length (bp) | References |
---|---|---|---|
COI | F- TCAACCAACCACAAAGACATTGGCAC | ~655 | [55] |
R- TAGACTTCTGGGTGGCCAAAGAATCA | |||
NADH2 | F- AAGGAGCAGTTTGATAGAGT | ~1050 | [40] |
R- AACGCTTAGCTGTTAATTAA |
Target Gene | Heat Activation | Denaturation | Annealing | Extension | Cycles | Final Extension |
---|---|---|---|---|---|---|
COI | 95 °C/15 min | 94 °C/30 s | 54 °C/50 s | 72 °C/60 s | 35 | 72 °C/10 min |
NADH2 | 95 °C /15 min | 94 °C/30 s | 48 °C/30 s | 72 °C/90 s | 35 | 72 °C/10 min |
Haplotype | COI | NADH2 |
---|---|---|
H1 | 1C_Pal (9 specimens) | 1N_Pal (9 specimens) |
H2 | 6C_Pal (4 specimens), 9C_Pal (4 specimens) | 6N_Pal (4 specimens), 9N_Pal (4 specimens) |
H3 | 7C_Pal (3 specimens) | 7N_Pal (3 specimens) |
H4 | 8C_Pal (6 specimens), 18C_Verd (5 specimens) | 8N_Pal (6 specimens), 18N_Verd (5 specimens) |
H5 | 12C_Pal (4 specimens), 32C_Gat (4 specimens) | 12N_Pal (4 specimens), 32N_Gat (4 specimens) |
H6 | 22C_Pal (3 specimens) | 22N_Pal (3 specimens) |
H7 | 24C_Pal (3 specimens) | 24N_Pal (3 specimens) |
H8 | 26C_Pal (3 specimens) | 26N_Pal (3 specimens) |
H9 | 30C_Pal (3 specimens) | 30N_Pal (3 specimens) |
H10 | 4C_Verd (6 specimens) | 4N_Verd (6 specimens) |
H11 | 15C_Verd (5 specimens), 16C_Verd (5 specimens), 25C_Verd (4 specimens), 33C_Spin (7 specimens) | 15N_Verd (5 specimens),33N_Spin (7 specimens) |
H12 | - | 16N_Verd (5 specimens), 25N_Verd (4 specimens) |
H13 | 17C_Verd (5 specimens), 3C_Spin (7 specimens) | 17N_Verd (5 specimens) |
H14 | - | 3N_Spin (7 specimens) |
H15 | 31C_Verd (5 specimens) | 31N_Verd (5 specimens) |
H16 | 2C_Gat (4specimens) | 2N_Gat (4 specimens) |
H17 | 10C_Gat (4 specimens) | 10N_Gat (4 specimens) |
H18 | 11C_Gat (4 specimens) | 11N_Gat (4 specimens) |
H19 | 19C_Gat (4 specimens) | 19N_Gat (4 specimens) |
H20 | 20C_Gat (4specimens) | 20N_Gat (4 specimens) |
H21 | 21C_Gat (4 specimens) | 21N_Gat (4 specimens) |
H22 | 5C_Spin (11 specimens) | 5N_Spin (11 specimens) |
Species | Number of Samples | Conservation Status | |||
---|---|---|---|---|---|
IUCN * | CITES | Barcelona Convention ** | Bern Convention | ||
Mustelus asterias | 21 | LC | -- | Annex III | -- |
Mustelus punctulatus | 20 | DD | -- | Annex III | -- |
Prionace glauca | 55 | CR | -- | Annex III | Appendix III |
Isurus oxyrinchus | 5 | CR | -- | Annex II | Appendix III |
Scyliorhinus canicula | 29 | LC | -- | -- | -- |
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Marchetti, P.; Mottola, A.; Piredda, R.; Ciccarese, G.; Di Pinto, A. Determining the Authenticity of Shark Meat Products by DNA Sequencing. Foods 2020, 9, 1194. https://doi.org/10.3390/foods9091194
Marchetti P, Mottola A, Piredda R, Ciccarese G, Di Pinto A. Determining the Authenticity of Shark Meat Products by DNA Sequencing. Foods. 2020; 9(9):1194. https://doi.org/10.3390/foods9091194
Chicago/Turabian StyleMarchetti, Patrizia, Anna Mottola, Roberta Piredda, Giuseppina Ciccarese, and Angela Di Pinto. 2020. "Determining the Authenticity of Shark Meat Products by DNA Sequencing" Foods 9, no. 9: 1194. https://doi.org/10.3390/foods9091194
APA StyleMarchetti, P., Mottola, A., Piredda, R., Ciccarese, G., & Di Pinto, A. (2020). Determining the Authenticity of Shark Meat Products by DNA Sequencing. Foods, 9(9), 1194. https://doi.org/10.3390/foods9091194