Molecular Genetic Methods for Species Identification of Milk: A Scoping Review

Introduction: Adulteration of dairy products, including the addition of cow’s milk to goat milk, remains a relevant issue affecting product authenticity, safety, and consumer protection. This type of adulteration is of particular concern due to the potential risks for individuals sensitive to cow’s milk proteins. Under these conditions, molecular genetic methods are considered a more promising approach for the detection of species adulteration in milk, as they are based on the analysis of species-specific DNA regions that are less susceptible to the effects of technological processing and biological factors compared with protein-based and physicochemical indicators.

Purpose: To conduct a comprehensive analysis of the current state of application of molecular genetic methods for the detection of species adulteration in dairy products in the context of preventing food fraud and ensuring high-quality products in the food industry.

Materials and Methods: The review was conducted in accordance with the PRISMA-ScR protocol. Publications were searched in Google Scholar, PubMed, and ScienceDirect using keywords focused on PCR analysis, DNA, goat and cow milk, and dairy product adulteration. The main inclusion criterion was the availability of data on the species identification of goat and cow milk using molecular genetic methods. During the analysis, information was extracted on the authors, year of publication, country of study, research object, animal species, methodology used, and key findings.

Results: A total of 7,437 records were identified through the screening process. After duplicate removal, 1,038 sources were retained for further analysis. During title and abstract screening, 937 publications that did not meet the inclusion criteria were excluded. Following full-text assessment of 101 articles, an additional 42 studies were excluded. Ultimately, 59 publications were included in the review. Analysis of the selected studies demonstrated that molecular genetic analytical methods are the most promising tool for species identification of milk and dairy products due to their high sensitivity and specificity. However, the effectiveness of detecting cow’s milk adulteration in goat milk largely depends on the type of sample matrix, the degree of technological processing, the selected genetic target, and the DNA amplification and detection method used. Real-time PCR and digital PCR demonstrated the highest sensitivity and reproducibility, whereas the lack of standardized approaches to target selection and analytical protocols remains a major limitation for the practical implementation of these methods in routine authenticity control of dairy products.

Conclusion: The main application of the findings of the present review may be the expansion of the scientific knowledge base on molecular genetic methods for confirming the authenticity of dairy product composition, providing approaches to address species adulteration and to protect consumer interests and health. The obtained data may facilitate the effective implementation of modern analytical methods for detecting cow’s milk adulteration in a wide range of products derived from non-traditional milk species. Practical implementation of the proposed approaches may contribute to improving the reliability of species identification of dairy raw materials, reducing the risk of economically motivated adulteration, and establishing reproducible protocols for testing laboratories, manufacturers, and regulatory authorities.

1. Abbas, O., Zadravec, M., Baeten, V., Mikuš, T., Lešić, T., Vulić, A., Prpić, J., Jemeršić, L., & Pleadin, J. (2018). Analytical methods used for the authentication of food of animal origin. Food Chemistry, 246, 6–17. https://doi.org/10.1016/j.foodchem.2017.11.007

2. Abedini, A., Salimi, M., Mazaheri, Y., Sadighara, P., Sani, M. A., Assadpour, E., & Jafari, S. M. (2023). Assessment of cheese frauds and relevant detection methods: A systematic review. Food Chemistry X, 100825. https://doi.org/10.1016/j.fochx.2023.100825

3. Agrimonti, C., Pirondini, A., Marmiroli, M., & Marmiroli, N. (2015). A quadruplex PCR (qxPCR) assay for adulteration in dairy products. Food Chemistry, 187, 58–64. https://doi.org/10.1016/j.foodchem.2015.04.017

4. Agustina, Y., Mulyo, J., Waluyati, L., & Mazwan, M. (2021). Consumer preferences toward goat milk. Agriecobis: Journal of Agricultural Socioeconomics and Business, 4(2), 100–109. https://doi.org/10.22219/agriecobis.v4i2.15922

5. Ait El Alia, O., Chaji, S., Boukrouh, S., Kaddouri, M., Zine-Eddine, Y., & Boutoial, K. (2026). Recent advances in the authentication of goat milk and its derived products. Journal of Food Composition and Analysis, 152, 109037. https://doi.org/10.1016/j.jfca.2026.109037

6. Artuvan, E., & Aksay, S. (2022). In food safety control overview of using real-time PCR. Biotech Studies, 31(2), 53–60. https://doi.org/10.38042/biotechstudies.1159145

7. Azad, T., & Ahmed, S. (2016). Common milk adulteration and their detection techniques. International Journal of Food Contamination, 3(1), 1–9. https://doi.org/10.1186/s40550-016-0045-3

8. Baptista, M., Cunha, J. T., & Domingues, L. (2021). DNA-based approaches for dairy products authentication: A review and perspectives. Trends in Food Science & Technology, 109, 386–397. https://doi.org/10.1016/j.tifs.2021.01.043

9. Bassi, C., Nuzzi, S., Pasinetti, S., Merlo, S., & Bello, V. (2026). Authenticity assessment of goat milk: Detecting dilution with cow milk by ML-enhanced speckle pattern imaging. Acta IMEKO, 15(1), 1–6. https://doi.org/10.21014/actaimeko.v15i1.2209

10. Böhme, K., Calo-Mata, P., Barros-Velázquez, J., & Ortea, I. (2019). Review of recent DNA-based methods for main food-authentication topics. Journal of Agricultural and Food Chemistry, 67(14), 3854–3864. https://doi.org/10.1021/acs.jafc.8b07016

11. Bougadi, E. T., & Kalogianni, D. P. (2020). DNA-based biosensor for food authenticity testing. Food Chemistry, 322, 126758. https://doi.org/10.1016/j.foodchem.2020.126758

12. Canja, C. M., Mazarel, A., Lupu, M. I., Padureanu, V., & Enache, D. V. (2016). Foodstuff falsification – a nowadays problem. Bulletin of the Transilvania University of Brasov. Series II, 9(2), 69–74.

13. Cavin, C., Cottenet, G., Blancpain, C., Bessaire, T., Frank, N., & Zbinden, P. (2016). Food adulteration: From vulnerability assessment to new analytical solutions. Chimia, 70(5), 329. https://doi.org/10.2533/chimia.2016.329

14. Choopan, R., Thanakiatkrai, P., & Kitpipit, T. (2017). Simultaneous species identification in milk and dairy products using direct PCR. Forensic Science International: Genetics Supplement Series, 6, e214–e215. https://doi.org/10.1016/j.fsigss.2017.09.077

15. Clark, S., & García, M. B. M. (2017). A 100-year review: Advances in goat milk research. Journal of Dairy Science, 100(12), 10026–10044. https://doi.org/10.3168/jds.2017-13287

16. Cosenza, G., Iannaccone, M., Gallo, D., & Pauciullo, A. (2019). A fast and reliable polymerase chain reaction method based on short interspersed nuclear elements detection for the discrimination of buffalo, cattle, goat, and sheep species in dairy products. Asian-Australasian Journal of Animal Sciences, 32(6), 891. https://doi.org/10.5713/ajas.18.0459

17. da Paixão Teixeira, J. L., dos Santos Caramês, E. T., Baptista, D. P., Gigante, M. L., & Pallone, J. A. L. (2021). Rapid adulteration detection of yogurt and cheese made from goat milk by vibrational spectroscopy and chemometric tools. Journal of Food Composition and Analysis, 96, 103712. https://doi.org/10.1016/j.jfca.2020.103712

18. da Silva Carvalho, A. C., Gennari, S. M., & Paschoalin, V. M. F. (2015). Consumption of animal products and frauds: DNA-based methods for the investigation of authenticity and traceability in dairy and meat-derived products – a review. Brazilian Journal of Veterinary Research and Animal Science, 52(3), 183–194. https://doi.org/10.11606/issn.1678-4456.v52i3p183-194

19. Danezis, G. P., Tsagkaris, A. S., Camin, F., Brusic, V., & Georgiou, C. A. (2016). Food authentication: Techniques, trends and emerging approaches. TrAC Trends in Analytical Chemistry, 85, 123–132. https://doi.org/10.1016/j.trac.2016.02.026

20. Deb, R., Sengar, G. S., Singh, U., Kumar, S., Raja, T. V., Alex, R., Alyethodi, R. R., & Prakash, B. (2017). LAMP assay for rapid diagnosis of cow DNA in goat milk and meat samples. Iranian Journal of Veterinary Research, 18(2), 134–137

21. Demiati, D., Wahyuni, W., Rafi, M., & Putra, B. (2023). The detection of goat milk adulteration with cow milk using a combination of voltammetric fingerprints and chemometrics analysis. Chemical Papers, 77. https://doi.org/10.1007/s11696-023-02780-w

22. Deng, L., Li, A., Gao, Y., Shen, T., Yue, H., Miao, J., Li, R., & Yang, J. (2020). Detection of the bovine milk adulterated in camel, horse, and goat milk using duplex PCR. Food Analytical Methods, 13, 560–567. https://doi.org/10.1007/s12161-019-01678-2

23. Di Domenico, M., Di Giuseppe, M., Rodríguez, J. W., & Cammà, C. (2017). Validation of a fast real-time PCR method to detect fraud and mislabeling in milk and dairy products. Journal of Dairy Science, 100(1), 106–112. https://doi.org/10.3168/jds.2016-11695

24. Di Febo, T., Schirone, M., Di Domenico, M., Visciano, P., Krasteva, I., Sonsini, L., Cerquaglia, O., Carpino, S., D’Onofrio, F., Tittarelli, M., & Luciani, M. (2020). Detection of undeclared bovine milk in different food matrices by a multi-technique approach. International Dairy Journal, 111, 104845. https://doi.org/10.1016/j.idairyj.2020.104845

25. Di Pinto, A., Terio, V., Marchetti, P., Bottaro, M., Mottola, A., Bozzo, G., Bonerba, E., Ceci, E., & Tantillo, G. (2017). DNA-based approach for species identification of goat-milk products. Food Chemistry, 229, 93–97. https://doi.org/10.1016/j.foodchem.2017.02.067

26. El Sheikha, A. F. (2019). DNAFoil: Novel technology for the rapid detection of food adulteration. Trends in Food Science & Technology, 86, 544–552. https://doi.org/10.1016/j.tifs.2018.11.012

27. Gallier, S., Tolenaars, L., & Prosser, C. (2020). Whole goat milk as a source of fat and milk fat globule membrane in infant formula. Nutrients, 12(11), 3486. https://doi.org/10.3390/nu12113486

28. Ganopoulos, I., Sakaridis, I., Argiriou, A., Madesis, P., & Tsaftaris, A. (2013). A novel closed-tube method based on high resolution melting (HRM) analysis for authenticity testing and quantitative detection in Greek PDO Feta cheese. Food Chemistry, 141(2), 835–840. https://doi.org/10.1016/j.foodchem.2013.02.130

29. Giglioti, R., Polli, H., Azevedo, B. T., Katiki, L. M., & Vercesi Filho, A. E. (2022). Detection and quantification of adulteration in milk and dairy products: A novel and sensitive qPCR-based method. Food Chemistry: Molecular Sciences, 4, 100074. https://doi.org/10.1016/j.fochms.2022.100074

30. Golinelli, L. P., Carvalho, A. C., Casaes, R. S., Lopes, C. S., Deliza, R., Paschoalin, V. M., & Silva, J. T. (2014). Sensory analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese. Journal of Dairy Science, 97(11), 6693–6699. https://doi.org/10.3168/jds.2014-7990

31. Guo, L., Qian, J. P., Guo, Y. S., Hai, X., Liu, G. Q., Luo, J. X., & Ya, M. (2018). Simultaneous identification of bovine and equine DNA in milks and dairy products inferred from triplex TaqMan real-time PCR technique. Journal of Dairy Science, 101(8), 6776–6786. https://doi.org/10.3168/jds.2018-14408

32. Guo, L., Ya, M., Hai, X., Guo, Y. S., Li, C. D., Xu, W. L., & Cai, Q. (2019). A simultaneous triplex TaqMan real-time PCR approach for authentication of caprine and bovine meat, milk and cheese. International Dairy Journal, 95, 58–64. https://doi.org/10.1016/j.idairyj.2019.03.004

33. Guo, L., Yu, Y., Xu, W. L., Li, C. D., Liu, G. Q., Qi, L., & Guo, Y. S. (2020). Simultaneous detection of ovine and caprine DNA in meat and dairy products using triplex TaqMan real-time PCR. Food Science & Nutrition, 8(12), 6467–6476. https://doi.org/10.1002/fsn3.1936

34. Habza-Kowalska, E., Grela, M., Gryzinska, M., & Listos, P. (2019). Molecular techniques for detecting food adulteration. Medycyna Weterynaryjna, 75(7), 404–409. https://doi.org/10.21521/mw.6261

35. Haddad, M., & Abu-Romman, S. (2020). PCR-based identification of bovine milk used in goat and sheep local dairy products marketed in Jordan. EurAsian Journal of BioSciences, 14(2), 5267–5272

36. Hadef, L., Hamad, B., & Bellabidi, M. (2024). A critical analysis of the impact of consumer attitudes and perception on goat milk consumption and influencing factors: consumer attitudes and perception towards goat milk. Pakistan Journal of Life and Social Sciences, 22. https://doi.org/10.57239/PJLSS-2024-22.2.00262

37. Hazra, T., Sharma, V., Sharma, R., & Arora, S. (2017). A species specific simplex polymerase chain reaction-based approach for detection of goat tallow in heat clarified milk fat (ghee). International Journal of Food Properties, 20(Suppl. 1), S69–S75. https://doi.org/10.1080/10942912.2017.1289542

38. Hazra, T., Sharma, V., Sharma, R., & Arora, S. (2018). PCR based assay for the detection of cow milk adulteration in buffalo milk. Indian Journal of Animal Research, 52(3), 383–387. https://doi.org/10.18805/ijar.11331

39. Kalogianni, D. P. (2018). DNA-based analytical methods for milk authentication. European Food Research and Technology, 244, 775–793. https://doi.org/10.1007/s00217-017-3016-x

40. Kang, T. S. (2019). Basic principles for developing real-time PCR methods used in food analysis: A review. Trends in Food Science & Technology, 91, 574–585. https://doi.org/10.1016/j.tifs.2019.07.037

41. Kashani, A., & Malau-Aduli, A. E. O. (2014). Real-time PCR and real-time RT-PCR applications in food labelling and gene expression studies. International Journal of Genetics and Genomics, 2, 6–12

42. Kastanos, E., Papaneophytou, C., Georgiou, T., & Demoliou, C. (2022). A simple and fast triplex-PCR for the identification of milk's animal origin in Halloumi cheese and yoghurt. Journal of Dairy Research, 89(3), 323–326. https://doi.org/10.1017/S0022029922000577

43. Khanzadi, S., Jamshidi, A., Razmyar, J., & Mohsenzadeh, M. (2014). PCR-based detection of cow and goat milk in sheep milk and dairy products marketed in Mashhad city of Iran. Iranian Journal of Veterinary Medicine, 7(4), 257–262

44. Kim, M. J., & Kim, H. Y. (2018). Direct duplex real-time loop mediated isothermal amplification assay for the simultaneous detection of cow and goat species origin of milk and yogurt products for field use. Food Chemistry, 246, 26–31. https://doi.org/10.1016/j.foodchem.2017.11.014

45. Klančnik, A., Toplak, N., Kovač, M., Ogrinc, N., & Jeršek, B. (2016). Robust PCR-based method for quantification of bovine milk in cheeses made from caprine and ovine milk. International Journal of Dairy Technology, 69(4), 540–549. https://doi.org/10.1111/1471-0307.12287

46. Lad, S. S., Aparnathi, K. D., Mehta, B., & Velpula, S. (2017). Goat milk in human nutrition and health: A review. International Journal of Current Microbiology and Applied Sciences, 6(6), 1781–1792. https://doi.org/10.20546/ijcmas.2017.605.194

47. Li, B., Yu, M., Xu, W., Chen, L., & Han, J. (2023). Comparison of PCR techniques in adulteration identification of dairy products. Agriculture, 13(7), 1450. https://doi.org/10.3390/agriculture13071450

48. Li, J. Y., Ge, Q., Liu, B., Gao, Z. P., & Guo, C. F. (2023). Quantification of cow milk in adulterated goat milk by HPLC-MS/MS using N-acetylglucosamine as a reliable biomarker of cow milk. Journal of Food Composition and Analysis, 123, 105583. https://doi.org/10.1016/j.jfca.2023.105583

49. Li, W., Huang, W., Fan, D., Gao, X., Zhang, X., Meng, Y., & Liu, T. C. (2023). Rapid quantification of goat milk adulteration with cow milk using Raman spectroscopy and chemometrics. Analytical Methods, 15(4), 455–461. https://doi.org/10.1039/d2ay01697d

50. Liao, J., Liu, Y. F., Ku, T., Liu, M. H., & Huang, Y. (2017). Qualitative and quantitative identification of adulteration of milk powder using DNA extracted with a novel method. Journal of Dairy Science, 100(3), 1657–1663. https://doi.org/10.3168/jds.2016-11900

51. Mafra, I., Honrado, M., & Amaral, J. S. (2022). Animal species authentication in dairy products. Foods, 11(8), 1124. https://doi.org/10.3390/foods11081124

52. Markiewicz-Keszycka, M., Czyżak-Runowska, G., Lipińska, P., & Wójtowski, J. (2013). Fatty acid profile of milk: A review. Bulletin of the Veterinary Institute in Pulawy, 57, 135. https://doi.org/10.2478/bvip-2013-0026

53. Osman, A. A., Mahmoud, A. M., & Soliman, A. S. (2014). Rapid detection of bovine-specific nucleic acid sequences in cow milk using polymerase chain reaction. International Journal of Nutrition and Food Sciences, 3(3), 141–144. https://doi.org/10.11648/j.ijnfs.20140303.12

54. Poonia, A., Jha, A., Sharma, R., Singh, H. B., Rai, A. K., & Sharma, N. (2017). Detection of adulteration in milk: A review. International Journal of Dairy Technology, 70(1), 23–42. https://doi.org/10.1111/1471-0307.12274

55. Reddy, D. M., Venkatesh, K., & Reddy, C. V. S. (2017). Adulteration of milk and its detection: A review. International Journal of Chemical Studies, 5(4), 613–617.

56. Rentsch, J., Weibel, S., Ruf, J., Eugster, A., Beck, K., & Köppel, R. (2013). Interlaboratory validation of two multiplex quantitative real-time PCR methods to determine species DNA of cow, sheep and goat as a measure of milk proportions in cheese. European Food Research and Technology, 236(1), 217–227. https://doi.org/10.1007/s00217-012-1880-y

57. Sangthong, D., Suwannarat, S., Samipak, S., & Sangthong, P. (2021). Multiplex PCR assay for species identification of meat and dairy products from buffalo (Bubalus bubalis), cattle (Bos indicus and Bos taurus), goat (Capra hircus), and sheep (Ovis aries). International Food Research Journal, 28(4). https://doi.org/10.47836/ifrj.28.4.08

58. Saviard, T., Deglaire, A., & Dupont, D. (2026). Seasonal variations in goat milk composition: A comprehensive review. International Dairy Journal, 176, 106569. https://doi.org/10.1016/j.idairyj.2026.106569

59. Seçkin, A. K., Yilmaz, B., & Tosun, H. (2017). Real-time PCR is a potential tool to determine the origin of milk used in cheese production. LWT, 77, 332–336. https://doi.org/10.1016/j.lwt.2016.11.065

60. Tăbăran, A., Mihaiu, M., Dan, S. D., Reget, O., Pivariu, B., Cordiş, I., & Mureşan, C. (2014). Identification of polymorphism in goat and sheep DGAT1 gene associated with milk production traits. Bulletin UASVM Veterinary Medicine, 71(2), 281–286. https://doi.org/10.15835/buasvmcn-vm:70:2:9325

61. Tortorici, L., Di Gerlando, R., Tolone, M., Mastrangelo, S., & Sardina, M. T. (2016). 12S rRNA mitochondrial gene as marker to trace Sicilian mono-species dairy products. Livestock Science, 193, 39–44. https://doi.org/10.1016/j.livsci.2016.09.015

62. Tsakali, E., Agkastra, C., Koliaki, C., Livanios, D., Boutris, G., Christopoulou, M. I., & Houhoula, D. (2019). Milk adulteration: Detection of bovine milk in caprine dairy products by real-time PCR. Journal of Food Research, 8(4), 52–57. https://doi.org/10.5539/jfr.v8n4p52

63. Tsiplakou, E., Kotrotsios, V., Hadjigeorgiou, I., & Zervas, G. (2010). Differences in sheep and goats milk fatty acid profile between conventional and organic farming systems. Journal of Dairy Research, 77, 343–349. https://doi.org/10.1017/S0022029910000270

64. Tsirigoti, E., Katsirma, Z., Papadopoulos, A. I., Samouris, G., Ekateriniadou, L. V., & Boukouvala, E. (2020). Application of triplex-PCR with an innovative combination of 3 pairs of primers for the detection of milk's animal origin in cheese and yoghurt. Journal of Dairy Research, 87(2), 239–242. https://doi.org/10.1017/S0022029920000242

65. Tuncay, R. M. (2023). Detection of adulteration of goat milk sold in the Turkish market by real-time polymerase chain reaction. German Journal of Veterinary Research, 3(1), 18–23. https://doi.org/10.51585/gjvr.2023.1.0048

66. Tuncay, R. M., & Sancak, Y. C. (2022). Comparison of PCR methods for determination of different types of milk added to goat milk. Balıkesir Sağlık Bilimleri Dergisi, 11(3), 509–514. https://doi.org/10.53424/balikesirsbd.1139179

67. Vafin, R. R., Galstyan, A. G., Tyulkin, S. V., Gilmanov, K. K., Yurova, E. A., Semipyatniy, V. K., & Bigaeva, A. V. (2022). Species identification of ruminant milk by genotyping of the κ-casein gene. Journal of Dairy Science, 105(2), 1004–1013. https://doi.org/10.3168/jds.2020-19931

68. Wang, Y., Jing, L., Li, Z., Li, C., Lu, Z., & Liu, L. (2021). Fluorescence-based PCR detection of goat milk powder mixed with cow milk. Asian Agricultural Research, 13, 43–48. https://doi.org/10.22004/ag.econ.317691

69. Wang, Y., Yu, Z., & Liu, Y. (2022). A high sensitivity method of closed-tube loop-mediated isothermal amplification developed for visual and rapid detection of cow milk adulteration. International Dairy Journal, 127, 105214. https://doi.org/10.1016/j.idairyj.2021.105214

70. Yadav, A. K., Gattupalli, M., Dashora, K., & Kumar, V. (2023). Key milk adulterants in India and their detection techniques: A review. Food Analytical Methods, 16(3), 499–514. https://doi.org/10.1007/s12161-022-02427-8

71. Yu, W., Chen, Y., Wang, Z., Qiao, L., Xie, R., Zhang, J., & Chen, A. (2021). Multiple authentications of high-value milk by centrifugal microfluidic chip-based real-time fluorescent LAMP. Food Chemistry, 351, 129348. https://doi.org/10.1016/j.foodchem.2021.129348

72. Zachar, P., Golian, J., Zaborska, D., Zacharová, A., & Mojžišová, J. (2011). Analytical methods for the species identification of milk and milk products. Mljekarstvo, 61(3), 199–207

73. Zengin, Z. S., & Kara, R. (2022). Investigation of milk origin in sheep and goat cheese with real-time PCR. Journal of Advances in VetBio Science and Techniques, 7(2), 179–184. https://doi.org/10.31797/vetbio.1057572

74. Zhang, X., Qiao, C., Fu, S., Jiao, Y., & Liu, Y. (2022). DNA-based qualitative and quantitative identification of bovine whey powder in goat dairy products. Journal of Dairy Science, 105(6), 4749–4759. https://doi.org/10.3168/jds.2021-21618

75. Zhizhin NA. Otsenka zhirnokislotnogo sostava korov'ego i koz'ego moloka s tochki zreniya funktsional'nogo vozdeystviya na organizm cheloveka. In: Sbornik nauchnykh trudov VNIMI. Moscow; 2020. p. 181–186. https://doi.org/10.37442/978-5-6043854-1-8-2020-1-181-186