European Food Research and Technology

, Volume 244, Issue 5, pp 775–793 | Cite as

DNA-based analytical methods for milk authentication

  • Despina P. Kalogianni
Review Article


Milk adulteration is a globally upcoming problem that concerns consumers, food industries and control authorities. Milk authenticity testing of dairy products is necessary, to protect consumers from fraudulent products, mislabeling and health risks and avoid unfair competition of food industries. Milk-based products produced by milk from specific animal origin are considered as healthier dairy products compared to others, leading to widespread acceptance of the consumers and food industries. This has made these animal-originated products extremely vulnerable to substitution with cheaper milk for economic profit. In addition, milk derived from specific animals (e.g., bovine) has been accused for allergenicity issues and other health problems, especially to sensitive human groups. Therefore, there is a great need for sensitive, accurate and quantitative analytical methods for milk adulteration detection in raw and processed milk-based products.


Nucleic acids DNA amplification Dairy products Milk Food authentication Food adulteration 


Compliance with ethical standards

Conflict of interest

The author declares that she has no conflict of interest.


  1. 1.
    Kamal M, Karoui R (2015) Analytical methods coupled with chemometric tools for determining the authenticity and detecting the adulteration of dairy products: a review. Trends Food Sci Technol 46:1–22CrossRefGoogle Scholar
  2. 2.
    Woolfe M, Primrose S (2004) Food forensics: using DNA technology to combat misdescription and fraud. Trends Biotechnol 22(5):222–226CrossRefGoogle Scholar
  3. 3.
    de la Fuente MA, Juárez M (2005) Authenticity assessment of dairy products. Crit Rev Food Sci Nutr 45:563–585CrossRefGoogle Scholar
  4. 4.
    Borková M, Snášelová J (2005) Possibilities of different animal milk detection in milk and dairy products—a review. Czech J Food Sci 23(2):41–50CrossRefGoogle Scholar
  5. 5.
    Jirillo F, Jirillo E, Magrone T (2010) Donkey’s and goat’s milk consumption and benefits to human health with special reference to the inflammatory status. Curr Pharm Des 16:859–863CrossRefGoogle Scholar
  6. 6.
    Zachar P, Šoltés M, Kasarda R, Novotný J, Novikmecová M, Marcinčáková D (2011) Analytical methods for the species identification of milk and milk products. Mljekarstvo 61(3):199–207Google Scholar
  7. 7.
    Poonia A, ALOK JHA, Sharma R, Singh HB, Rai AK, Sharma N (2017) Detection of adulteration in milk: a review. Int J Dairy Technol 70:23–42CrossRefGoogle Scholar
  8. 8.
    Soller L, Mill C, Avinashi V, Teoh T, Edmond S. Chan ES (2017) Development of anaphylactic cow’s milk allergy following cow’s milk elimination for eosinophilic esophagitis in a teenager. J Allergy Clin Immunol Pract (Article in Press) Google Scholar
  9. 9.
    Caira S, Nicolai MA, Lilla S, Calabrese MG, Pinto G, Scaloni A, Chianese L, Addeo F (2017) Eventual limits of the current EU official method for evaluating milk adulteration of water buffalo dairy products and potential proteomic solutions. Food Chem 230:482–490CrossRefGoogle Scholar
  10. 10.
    Brandao MP, Neto MG, de Carvalho dos Anjos V, Bell MGV (2017) Detection of adulteration of goat milk powder with bovine milk powder by front-face and time resolved fluorescence. Food Control 81:168–172CrossRefGoogle Scholar
  11. 11.
    Moore JC, Spink J, Lipp M (2012) Development and application of a database of food ingredient fraud and economically motivated adulteration from 1980 to 2010. J Food Sci 77(4):118–126CrossRefGoogle Scholar
  12. 12.
    Mayer HK (2005) Milk species identification in cheese varieties using electrophoretic, chromatographic and PCR techniques. Int Dairy J 15:595–604CrossRefGoogle Scholar
  13. 13.
    Rentsch J, Weibel S, 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. Eur Food Res Technol 236:217–227CrossRefGoogle Scholar
  14. 14.
    Bartuzi Z, Cocco RR, Muraro A, Nowak-Węgrzyn A (2017) Contribution of molecular allergen analysis in diagnosis of milk allergy. Curr Allergy Asthma Rep 17(46):1–9Google Scholar
  15. 15.
    Hurley IP, Ireland HE, Coleman RC, Williams JHH (2004) Application of immunological methods for the detection of species adulteration in dairy products. Int J Food Sci Technol 39:873–878CrossRefGoogle Scholar
  16. 16.
    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 Chem 141:835–840CrossRefGoogle Scholar
  17. 17.
    De S, Brahma B, Polley S, Mukherjee A, Banerjee D, Gohaina M, Singh KP, Singh R, Datta TK, Goswami SL (2011) Simplex and duplex PCR assays for species specific identification of cattle and buffalo milk and cheese. Food Control 22:690–696CrossRefGoogle Scholar
  18. 18.
    Ali E, Bee S, Hamid A, Hossain MAM, Mustafa S, Kader A, Zaidul ISM (2016) Lab-on-a-Chip-based PCR-RFLP assay for the detection of Malayan box turtle (Cuora amboinensis) in the food chain and traditional Chinese medicines. PloS One 11(10):1–27Google Scholar
  19. 19.
    Di Rienzo V, Miazzi MM, Fanelli V, Savino V, Pollastro S, Colucci F, Miccolupo A et al (2016) An enhanced analytical procedure to discover table grape DNA adulteration in industrial musts. Food Control 60:124–130CrossRefGoogle Scholar
  20. 20.
    Fajardo V, González I, Rojas M, García T, Martín R (2010) A review of current methodologies for the authentication of meats from game animal species. Trends Food Sci Technol 21:408–421CrossRefGoogle Scholar
  21. 21.
    Plath A, Krause I, Einspanier R (1997) Species identification in dairy products by three different DNA-based techniques. Z Lebensm Unters Forsch 205:437–441CrossRefGoogle Scholar
  22. 22.
    Lanzilao I, Burgalassi F, Fancelli S, Settimelli M, Fani R (2005) Polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial cytb gene from species of dairy interest. J AOAC Int 88:128–135Google Scholar
  23. 23.
    Abdel-Rahman SM, Ahmed MMM (2007) Rapid and sensitive identification of buffalo’s, cattle’s and sheep’s milk using species-specific PCR and PCR–RFLP techniques. Food Control 18:1246–1249CrossRefGoogle Scholar
  24. 24.
    Otaviano AR, Lima ALF, Laureano MMM, Sena JAD, De Albuquerque LG, Tonhati H (2008) β-casein gene polymorphism permits identification of bovine milk mixed with bubaline milk in mozzarella cheese. Genet Mol Biol 31(4):902–905CrossRefGoogle Scholar
  25. 25.
    Maudet LIA, Taberlet P, Ce B, De Biologie L, Umr C, Universite T, Fourier J, Grenoble F, France C (2001) Detection of cow’s milk in goat’s cheeses inferred from mitochondrial DNA polymorphism. J Dairy Res 68:229–235CrossRefGoogle Scholar
  26. 26.
    Bania BJ, Ugorski M, Polanowski A (2001) Application of polymerase chain reaction for detection of goat’s milk adulteration by milk of cow. J Dairy Sci 68:333–336Google Scholar
  27. 27.
    Rea BS, Chikuni K, Branciari R, Sangamayya RAMS., Ranucci D, Avellini P (2001) Use of duplex polymerase chain reaction (duplex-PCR) technique to identify bovine and water buffalo milk used in making mozzarella cheese. J Dairy Res 68:689–698CrossRefGoogle Scholar
  28. 28.
    Mašková E, Paulíčková I (2006) PCR-based detection of cow’s milk in goat and sheep cheeses marketed in the Czech Republic. Czech J Food Sci 24:127–132CrossRefGoogle Scholar
  29. 29.
    Bottero MT, Civera T, Nucera D, Rosati S, Sacchi P, Turi RM (2003) A multiplex polymerase chain reaction for the identification of cow’s, goat’s and sheep’s milk in dairy products. Int Dairy J 13:277–282CrossRefGoogle Scholar
  30. 30.
    López-Calleja I, González Alonso I, Fajardo V, Rodríguez MA, Hernández PE, García T, Martín R (2004) Rapid detection of cow’s milk in sheep’s and goat’s milk by a species-specific polymerase chain reaction technique. J Dairy Sci 87:2839–2845CrossRefGoogle Scholar
  31. 31.
    López-Calleja I, González Alonso I, Fajardo V, Rodríguez MA, Hernández PE, García T, Martín R (2005) PCR detection of cows’ milk in water buffalo milk and mozzarella cheese. Int Dairy J 15:1122–1129CrossRefGoogle Scholar
  32. 32.
    López-Calleja I, González I, Fajardo V, Martín I, Hernández PE, García T, Martín R (2005) Application of polymerase chain reaction to detect adulteration of sheep’s milk with goats’ milk. J Dairy Sci 88:3115–3120CrossRefGoogle Scholar
  33. 33.
    Díaz ILC, Alonso IG, Fajardo V, Martín I, Hernández P, Lacarra TG, De Santos RM (2007) Application of a polymerase chain reaction to detect adulteration of ovine cheeses with caprine milk. Eur Food Res Technol 225:345–349CrossRefGoogle Scholar
  34. 34.
    Mafra I, Roxo Á, Ferreira IMPLVO., Oliveira MBPP. (2007) A duplex polymerase chain reaction for the quantitative detection of cows’ milk in goats’ milk cheese. Int Dairy J 17:1132–1138CrossRefGoogle Scholar
  35. 35.
    Rodrigues NPA, Givisiez PEN, Queiroga RCRE., Azevedo PS, Gebreyes WA (2012) Milk adulteration: detection of bovine milk in bulk goat milk produced by smallholders in northeastern Brazil by a duplex PCR assay. J Dairy Sci 95:2749–2752CrossRefGoogle Scholar
  36. 36.
    Golinelli LP, Carvalho AC, Casaes RS, Lopes CSC, Deliza R, Paschoalin VMF, Silva JT (2014) Sensory analysis and species-specific PCR detect bovine milk adulteration of frescal (fresh) goat cheese. J Dairy Sci 97:6693–6699CrossRefGoogle Scholar
  37. 37.
    Tortorici L, Di Gerlando R, Tolone M, Mastrangelo S, Sardina MT (2016) Dairy products. Livest Sci 193:39–44CrossRefGoogle Scholar
  38. 38.
    Di Pinto A, Conversano MC, Forte VT, Novello L, Tantillo GM (2004) Detection of cow milk in buffalo “mozzarella” by polymerase chain reaction (PCR) assay. J Food Qual 27:428–435CrossRefGoogle Scholar
  39. 39.
    Feligini M, Bonizzi I, Curik VC, Parma P, Greppi GF, Enne G (2005) Detection of adulteration in Italian Mozzarella cheese using mitochondrial DNA templates as biomarkers. Food Technol Biotechnol 43:91–95Google Scholar
  40. 40.
    Kotowicz M, Adamczyk E, Bania J (2007) Application of a duplex-PCR for detection of cow’s milk in goat’s milk. Ann Agric Environ Med 14:215–218Google Scholar
  41. 41.
    Bai WL, Yin RH, Zhao SJ, Dou QL, Yang JC, Jiang WQ, Zhao ZH, Luo GB (2009) Rapid detection of bovine milk in yak milk using a polymerase chain reaction technique. J Dairy Sci 92:1354–1360CrossRefGoogle Scholar
  42. 42.
    Reale S, Campanella A, Merigioli A, Pilla F (2008) A novel method for species identification in milk and milk-based products. J Dairy Res 75:107–112CrossRefGoogle Scholar
  43. 43.
    Gonçalves J, Pereira F, Amorim A, van Asch B (2012) New method for the simultaneous identification of cow, sheep, goat, and water buffalo in dairy products by analysis of short species-specific mitochondrial DNA targets. J Agric Food Chem 60:10480–10485CrossRefGoogle Scholar
  44. 44.
    Liao J, Liu YF, Ku T, Liu MH, Huang Y (2017) Qualitative and quantitative adulteration identification of milk powder using the DNA with novel extraction method. J Dairy Sci 100:1–7CrossRefGoogle Scholar
  45. 45.
    López-Calleja I, González I, Fajardo V, Martín I, Hernández PE, García T, Martín R (2007) Quantitative detection of goats’ milk in sheep’s milk by real-time PCR. Food Control 18:1466–1473CrossRefGoogle Scholar
  46. 46.
    Zhang C, Fowler MR, Scott NW, Lawson G, Slater A (2007) A TaqMan real-time PCR system for the identification and quantification of bovine DNA in meats, milks and cheeses. Food Control 18:1149–1158CrossRefGoogle Scholar
  47. 47.
    Cottenet G, Blancpain C, Golay P (2011) Simultaneous detection of cow and buffalo species in milk from China, India and Pakistan using multiplex real-time PCR. J Dairy Sci 94:3787–3793CrossRefGoogle Scholar
  48. 48.
    Dalmasso A, Civera T, La Neve F, Bottero MT (2011) Simultaneous detection of cow and buffalo milk in mozzarella cheese by Real-Time PCR assay. Food Chem 124:362–366CrossRefGoogle Scholar
  49. 49.
    Lopparelli RM, Cardazzo B, Balzan S, Giaccone V, Novelli E (2007) Real-Time TaqMan polymerase chain reaction detection and quantification of cow DNA in pure water buffalo mozzarella cheese: method validation and its application on commercial samples. J Agric Food Chem 55:3429–3434CrossRefGoogle Scholar
  50. 50.
    Drummond MG, Brasil BSAF., Dalsecco LS, Brasil RSAF., Teixeira LV, Oliveira DAA (2013) A versatile real-time PCR method to quantify bovine contamination in buffalo products. Food Control 29:131–137CrossRefGoogle Scholar
  51. 51.
    Domenico M, Di Giuseppe M, Di Rodríguez JDW, Cammà C (2017) Validation of a fast real-time PCR method to detect fraud and mislabeling in milk and dairy products. J Dairy Sci 100:106–112CrossRefGoogle Scholar
  52. 52.
    Sakaridis I, Ganopoulos I, Argiriou A, Tsaftaris A (2013) High resolution melting analysis for quantitative detection of bovine milk in pure water buffalo mozzarella and other buffalo dairy products. Int Dairy J 28:32–35CrossRefGoogle Scholar
  53. 53.
    Agrimonti C, Pirondini A, Marmiroli M, Marmiroli N (2015) A quadruplex PCR (qxPCR) assay for adulteration in dairy products. Food Chem 187:58–64CrossRefGoogle Scholar
  54. 54.
    Kounelli ML, Kalogianni DP (2017) A sensitive DNA based fluorometric method for milk authenticity of dairy products based on spectrally distinct microspheres. Eur Food Res Technol (Article in press) Google Scholar
  55. 55.
    Beltramo C, Riina MV, Colussi S, Campia V, Maniaci MG, Biolatti C, Trisorio S, Modesto P, Peletto S, Acutis PL (2017) Validation of a DNA biochip for species identification in food forensic science. Food Control 78:366–373CrossRefGoogle Scholar
  56. 56.
    Littlefair JE, Clare EL (2016) Barcoding the food chain: from Sanger to high-throughput sequencing. Genome 59:946–958CrossRefGoogle Scholar
  57. 57.
    Pečnikar ŽF, Buzan EV (2014) 20 years since the introduction of DNA barcoding: from theory to application. J Appl Genet 55:43–52CrossRefGoogle Scholar
  58. 58.
    Liu Y, Xiang L, Zhang Y, Lai X, Xiong C, Li J, Su Y, Sun W, Chen S (2018) DNA barcoding based identification of Hippophae species and authentication of commercial products by high resolution melting analysis. Food Chem 242:62–67CrossRefGoogle Scholar
  59. 59.
    Bosmali I, Ordoudi SA, Tsimidou MZ, Madesis P (2017) Greek PDO saffron authentication studies using species specific molecular markers. Food Res Int 100(1):899–907CrossRefGoogle Scholar
  60. 60.
    Parveen I, Gafner S, Techen N, Murch SJ, Khan IA (2016) DNA Barcoding for the identification of botanicals in herbal medicine and dietary supplements: strengths and limitations. Planta Med 82(14):1225–1235CrossRefGoogle Scholar
  61. 61.
    Mishra P, Kumar A, Nagireddy A, Mani DN, Shukla AK, Tiwari R, Sundaresan V (2016) DNA barcoding: an efficient tool to overcome authentication challenges in the herbal market. Plant Biotechnol J 14(1):8–21CrossRefGoogle Scholar
  62. 62.
    Paracchini V, Petrillo M, Lievens A, Puertas Gallardo A, Martinsohn JT, Hofherr J, Maquet A, Silva APB, Kagkli DM, Querci M, Patak A, Angers-Loustau (2017) A novel nuclear barcode regions for the identification of flatfish species. Food Control 79:297–308CrossRefGoogle Scholar
  63. 63.
    Fernandes TJ, Costa J, Oliveira MB, Mafra I (2017) DNA barcoding coupled to HRM analysis as a new and simple tool for the authentication of Gadidae fish species. Food Chem 230:49–57CrossRefGoogle Scholar
  64. 64.
    Ratnasingham S, Hebert PDN (2007) BOLD: The Barcode of Life Data System. Mol Ecol Notes 7(3):355–364. http://www.barcodinglife.orgGoogle Scholar
  65. 65.
    Sarwat M, Yamdagni MM (2016) DNA barcoding, microarrays and next generation sequencing: recent tools for genetic diversity estimation and authentication of medicinal plants. Crit Rev Biotechnol 36(2):191–203CrossRefGoogle Scholar
  66. 66.
    Burns M, Wiseman G, Knight A, Bramley P, Foster L, Rollinson S, Damant A, Primrose S (2016) Measurement issues associated with quantitative molecular biology analysis of complex food matrices for the detection of food fraud. Analyst 141:45–61CrossRefGoogle Scholar
  67. 67.
    Tillmar AO, Dell’Amico B, Welander J, Holmlund G (2013) A universal method for species identification of mammals utilizing next generation sequencing for the analysis of DNA mixtures. PLoS One 8(12):e83761CrossRefGoogle Scholar
  68. 68.
    Yang T, Bao SY, Ford R, Jia TJ, Guan JP, He YH, Sun XL, Jiang JY, Hao JJ, Zhang XY, Zong XX (2012) High-throughput novel microsatellite marker of faba bean via next generation sequencing. BMC Genom 13:602–612CrossRefGoogle Scholar
  69. 69.
    Bertolini F, Ghionda MC, D’Alessandro E, Geraci C, Chiofalo V, Fontanesi L (2015) A next generation semiconductor based sequencing approach for the identification of meat species in DNA mixtures. PLoS One 10(7):e0131925CrossRefGoogle Scholar
  70. 70.
    Kappel K, Haase I, Käppel C, Sotelo CG, Schröder U (2017) Species identification in mixed tuna samples with next-generation sequencing targeting two short cytochrome b gene fragments. Food Chem 234:212–219CrossRefGoogle Scholar
  71. 71.
    Li JJ, Xiong C, Liu Y, Liang JS, Zhou XW (2016) Loop-mediated isothermal amplification (LAMP): emergence as an alternative technology for herbal medicine identification. Front Plant Sci 7:1956–1966Google Scholar
  72. 72.
    Zhao Y, Chen F, Li Q, Wang L, Fan C (2015) Isothermal amplification of nucleic acids. Chem Rev 115:12491 – 12545CrossRefGoogle Scholar
  73. 73.
    Lee MS, Su TY, Lien YY, Sheu SC (2017) The development of loop-mediated isothermal amplification (LAMP) assays for the rapid authentication of five forbidden vegetables in strict vegetarian diets. Sci Rep 7:44238-CrossRefGoogle Scholar
  74. 74.
    Abdulmawjood A, Grabowski N, Fohler S, Kittler S, Nagengast H, Klein G (2014) Development of loop-mediated isothermal amplification (LAMP) assay for rapid and sensitive identification of ostrich meat. PLoS One 9(6):e100717CrossRefGoogle Scholar
  75. 75.
    Li M, Wong YL, Jiang LL, Wong KL, Wong YT, Lau CBS, Shaw PC (2013) Application of novel loop-mediated isothermal amplification (LAMP) for rapid authentication of the herbal tea ingredient Hedyotis diffusa Willd. Food Chem 141:2522–2525CrossRefGoogle Scholar
  76. 76.
    Cao L, Cui X, Hu J, Li Z, Choi JR, Yang Q, Lin M, HuiLi Y, Xu F (2017) Advances in digital polymerase chain reaction (dPCR) and its emerging biomedical applications. Biosens Bioelectron 90:459–474CrossRefGoogle Scholar
  77. 77.
    Shehata HR, Li J, Chen S, Redda H, Cheng S, Tabujara N, Li H, Warriner K, Hanne R (2017) Droplet digital polymerase chain reaction (ddPCR) assays integrated with an internal control for quantification of bovine, porcine, chicken and turkey species in food and feed. PLoS One 12(8):e0182872CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2017

Authors and Affiliations

  1. 1.Department of ChemistryUniversity of PatrasPatrasGreece

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