Development of multiplex PCR for species-specific DNA determination of saury (Cololabis saira) and far eastern sardine, ivasi (Sardinops melanostictus)
https://doi.org/10.21323/2618-9771-2025-8-3-324-334
Abstract
The paper presents the development and validation of a multiplex real-time polymerase chain reaction (real-time PCR) method forspecies identification of canned products from saury (Cololabis saira) and Far Eastern sardine (Ivasi) (Sardinops melanostictus). The method is based on the use of the oligonucleotide system «Saury-Ivasi-VKO» and is aimed at solving the problem of adulteration of fish products, which is widespread in the Russian market. To assess the specificity of the method, a control panel of 90 fish species and 195 food fish samples were used. The method demonstrated high sensitivity, allowing detection of DNA of target species at up to 0.01% in the sample. The specificity of primers was confirmed by the absence of DNA amplification of unrelated species. The conducted experiments showed that the declared fish species were correctly identified in 100% of cases, and there were no false negative results. The results of food product testing revealed nonconformities in 34% of canned saury samples, of which 21 % were replaced by sardine and 6% by Pacific herring. The developed methodology reduces the cost of analysis due to the lack of dependence on imported reagents and can be implemented in laboratories working with GMOs and species identification. The conclusions confirm the effectiveness of the proposed approach for quality control and prevention of adulteration of fish products.
Keywords
Supporting agencies: The article was published as part of the research topic No. FGUS-2025-0009 of the state assignment of the V. M. Gorbatov Federal Research Center for Food Systems of RAS.
About the Authors
T. A. Fomina
V. M. Gorbatov Federal Research Center for Food Systems; Nation Center for Fish and Agricultural Product Safety
Russian Federation
Russian Federation
Tatyana A. Fomina, Candidate of Technical Sciences, Senior Research Scientist, Laboratory of Molecular Biology and Bioinformatics; Head of the Department of Molecular Diagnostic Research at the Testing Reference Laboratory
26, Talalikhin str., 109316, Moscow; 14–1, Grafskiy per., 129626, Moscow
D. V. Khvostov
V. M. Gorbatov Federal Research Center for Food Systems
Russian Federation
Russian Federation
Daniil V. Khvostov, Candidate of Technical Sciences, Scientific Worker, Laboratory of Molecular Biology and Dioinformatics
26, Talalikhin str., 109316, Moscow
M. Yu. Minaev
V. M. Gorbatov Federal Research Center for Food Systems
Russian Federation
Russian Federation
Mikhail Yu. Minaev, Candidate of Technical Sciences, Head of Laboratory of Molecular Biology and Bioinformatics
26, Talalikhin str., 109316, Moscow
D. A. Utyanov
V. M. Gorbatov Federal Research Center for Food Systems
Russian Federation
Russian Federation
Dmitry A. Utyanov, Candidate of Technical Sciences, Scientific Worker, Laboratory of Scientific and Methodical Work, Biological and Analytical Research
26, Talalikhin str., 109316, Moscow
References
1. Suyama, S., Shimizu, A., Isu, S., Ozawa, H., Morioka, T., Nakaya, M. et al. (2016). Determination of the spawning history of Pacific saury Cololabis saira from rearing experiments: Identification of post-spawning fish from histological observations of ovarian arterioles. Fisheries Science, 82(3), 445–457. https://doi.org/10.1007/s12562-016-0980-1
2. Antonenko, D. V. (2023). Russian saury fishery and factors influencing its distribution in the Northwest Pacific Ocean. Trudy VNIRO, 194, 108–117. (In Russian) https://doi.org/10.36038/2307-3497-2023-194-108-117
3. Ustinova, E. I., Filatov, V. N., Chulchekov, D. N. (September 5–9, 2022). Changes in the oceanological conditions and their effect on spatial movement of commercial concentrations of Pacific saury, sardine and mackerel in the NorthWestern part of the Pacific Ocean. Development of the main transport waterways under the conditions of the global climate change on the territory of the Russian federation (Eurasia) (“Hazardous phenomena — IV”). Rostov-on-Don: SSC RAS. 2022.
4. Xing, Q., Yu, H., Liu, Y., Li, J., Tian, Y., Bakun, A. et al. (2022). Application of a fish habitat model considering mesoscale oceanographic features in evaluating climatic impact on distribution and abundance of Pacific saury (Cololabis saira). Progress in Oceanography, 201, Article 102743. https://doi.org/10.1016/j.pocean.2022.102743
5. Sarr, O., Kindong, R., Tian, S. (2021). Knowledge on the Biological and Fisheries Aspects of the Japanese Sardine, Sardinops melanostictus (Schlegel, 1846). Journal of Marine Science and Engineering, 9(12), Article 1403. https://doi.org/10.3390/jmse9121403
6. Takasuka, A., Oozeki, Y., Kubota, H., Lluch-Cota, S. E. (2008). Contrasting spawning temperature optima: Why are anchovy and sardine regime shifts synchronous across the North Pacific? Progress in Oceanography, 77(2–3), 225–232. https://doi.org/10.1016/j.pocean.2008.03.008
7. Do, T. D., Wong, L. L. (2025). The investigation of seafood mislabeling in Asia: A review and coping strategies. Journal of Agriculture and Food Research, Article 101702. https://doi.org/10.1016/j.jafr.2025.101702
8. Lobacev, D. (2023). And yet it is sardine. What kind of fish is ivasi and what it is eaten with. Retrieved from https://tass.ru/obschestvo/18253477 Accessed May 12, 2025. (In Russian)/
9. Fanelli, V., Mascio, I., Miazzi, M. M., Savoia, M. A., De Giovanni, C., Montemurro, C. (2021). Molecular approaches to agri-food traceability and authentication: An updated review. Foods, 10(7), Article 1644. https://doi.org/10.3390/foods10071644
10. Galimberti, A., Casiraghi, M., Bruni, I., Guzzetti, L., Cortis, P., Berterame, N. M. et al. (2019). From DNA barcoding to personalized nutrition: The evolution of food traceability. Current Opinion in Food Science, 28, 41–48. https://doi.org/10.1016/j.cofs.2019.07.008
11. Armani, A., Castigliego, L., Tinacci, L., Gianfaldoni, D., Guidi, A. (2012). Multiplex conventional and real-time PCR for fish species identification of Bianchetto (juvenile Sardina pilchardus), Rossetto (Aphia minuta), and icefish in fresh, marinated and cooked products. Food Chemistry, 133(1), 184–192. https://doi.org/10.1016/j.foodchem.2011.12.076
12. Murray, M. G., Thompson, W. F. (1980). Rapid isolation of high molecular weight plant DNA. Nucleic Acids Research, 8(19), 4321–4325. https://doi.org/10.1093/nar/8.19.4321
13. Boom, R., Sol, C. J., Salimans, M. M. M., Jansen, C. L., Wertheim-Van Dillen, P. M., Van Der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. Journal of Clinical Microbiology, 28(3), 495–503. https://doi.org/10.1128/jcm.28.3.495-503.1990
14. Cutarelli, A., Galiero, G., Capuano, F., Corrado, F. (2018). Species identification by means of mitochondrial cytochrome b DNA sequencing in processed anchovy, sardine and tuna products. Food and Nutrition Sciences, 9(4), 369–375. https://doi.org/10.4236/fns.2018.94029
15. Ramos, A., Santos, C., Mateiu, L., Gonzalez, M. del M., Alvarez, L., Azevedo, L. et al. (2013). Frequency and pattern of heteroplasmy in the complete human mitochondrial genome. PLoS ONE, 8(10), Article e74636. https://doi.org/10.1371/journal.pone.0074636
16. National Center for Biotechnology Information (NCBI). Retrieved from https://www.ncbi.nlm.nih.gov/ Accessed June 13, 2025.
17. Basic Local Alignment Search Tool (BLAST). Retrieved from https://blast.ncbi. nlm.nih.gov/Blast.cgi Accessed June 13, 2025.
18. Integrated DNA Technologies. Web service for determining the physical properties of oligonucleotides OligoAnalyzer. Retrieved from https://eu.idtdna.com/pages/tools/oligoanalyzer Accessed June 13, 2025.
19. WHO. (2022). WHO global strategy for food safety 2022–2030: Towards stronger food safety systems and global cooperation. Geneva 27, Switzerland, 2022.
20. Revealed: Seafood fraud happening on a vast global scale. Retrieved from https://www.theguardian.com/environment/2021/mar/15/revealed-seafoodhappening-on-a-vast-global-scale Accessed June 13, 2025.
21. Luque, G. M., Donlan, C. J. (2019). The characterization of seafood mislabeling: A global meta-analysis. Biological Conservation, 236, 556–570. https://doi.org/10.1016/j.biocon.2019.04.006
22. Timoshkina, N. N., Vodolazhskii, D. I., Usatov, A. V. (2011). Molecular-genetic markers in studies of intra- and interspecies polymorphism in sturgeon (acipenseriformes). Russian Journal of Genetics: Applied Research, 1(2), 160–171. https://doi.org/10.1134/S2079059711020122
23. Mariani, S., Griffiths, A. M., Velasco, A., Kappel, K., Jérôme, M., Perez-Martin, R. I. et al. (2015). Low mislabeling rates indicate marked improvements in European seafood market operations. Frontiers in Ecology and the Environment, 13(10), 536–540. https://doi.org/10.1890/150119
24. Xiong, X., Yuan, F., Huang, M., Cao, M., Xiong, X. (2020), Development of a rapid method for codfish identification in processed fish products based on SYBR Green real-time PCR. International Journal of Food Science and Technology, 55(4), 1843–1850. https://doi.org/10.1111/ijfs.14446
25. Seafood Fraud Becoming a Costly Global Concern. Retrieved from https://foodinstitute.com/focus/seafood-fraud-becoming-a-costly-global-concern Accessed June 13, 2025.
Review
For citations:
Fomina T.A., Khvostov D.V., Minaev M.Yu., Utyanov D.A. Development of multiplex PCR for species-specific DNA determination of saury (Cololabis saira) and far eastern sardine, ivasi (Sardinops melanostictus). Food systems. 2025;8(3):324-334. (In Russ.) https://doi.org/10.21323/2618-9771-2025-8-3-324-334
Views:
36
Email this article 