Authentication of caprine milk and cheese by commercial qPCR assay
Keywords:bovine, caprine, adulteration, qPCR
The objective of the study was to investigate potential adulteration of commercial caprine milks and cheeses with bovine milk using commercial qPCR assay. The assay comprised of bovine-, ovine- and caprine-specific primers and TaqMan probe and mammalian internal control. Specificity, sensitivity, linearity, reproducibility and efficiency of the bovine assay were tested as well. Specificity was verified by running reaction on the DNA of other milk-producing species (caprine and ovine) and made-up bovine-caprine (v/v) milk mixes. In both experiments, a bovine DNA fragment was amplified whereas no amplification was obtained from the other species. Sensitivity, linearity, reproducibility and efficiency were tested on 10-fold dilution series of 10 ng bovine DNA. The assay has shown good linearity (R2 = 0.983) within whole range, with efficiency of 86% and excellent reproducibility (SD around the CT for the technical replicates <0.5). The sensitivity was adequate, as calculated LOD and LOQ were 1.44 pg and 2.94 pg of bovine DNA, respectively. Finally, the assay was used to authenticate 5 caprine milk samples and 5 caprine cheese samples, purchased from local supermarkets. Totally, 1 milk sample has shown the fluorescence signal, which exceeded baseline in cycle 39.01 ±0.69. However, the signal was above LOD and LOQ suggesting that there could not be unambiguously declared any adulteration with bovine milk. Amplification of bovine-specific DNA was not observed in the other samples indicating products were not adulterated. The commercial qPCR assay has proved that real-time PCR assays, as well as DNA-based techniques in a general, are the excellent and reliable tools for fighting with frauds in the food industry and protecting the public health.
Agrimonti, C., Pirondini, A., Marmiroli, M., Marmiroli, N. 2015. A quadruplex PCR (qxPCR) assay for adulteration in dairy products. Food Chemistry, vol. 187, p. 58-64. https://doi.org/10.1016/j.foodchem.2015.04.017
Azad, T., Ahmed, S. 2016. Common milk adulteration and their detection techniques. International Journal Of Food Contamination, vol. 3, no. 1, p. 22. https://doi.org/10.1186/s40550-016-0045-3
Barłowska, J., Szwajkowska, M., Litwińczuk, Z., Król, J. 2011. Nutritional Value and Technological Suitability of Milk from Various Animal Species Used for Dairy Production. Comprehensive Reviews In Food Science And Food Safety, vol. 10, no. 6, p. 291-302. https://doi.org/10.1111/j.1541-4337.2011.00163.x
Bottero, M., Civera, T., Nucera, D., Rosati, S., Sacchi, P., Turi, R. 2003. A multiplex polymerase chain reaction for the identification of cows', goats' and sheep's milk in dairy products. International Dairy Journal, vol. 13, no. 4, p. 277-282. https://doi.org/10.1016/S0958-6946(02)00170-X
Branciari, R., Nijman, I., Plas, M., Di Antonio, E., Lenstra, J. 2000. Species Origin of Milk in Italian Mozzarella and Greek Feta Cheese. Journal Of Food Protection, vol. 63, no. 3, p. 408-411. https://doi.org/10.4315/0362-028X-63.3.408
Caldwell, J. 2017. Food Analysis Using Organelle DNA and the Effects of Processing on Assays. Annual Review Of Food Science And Technology, vol. 8, no. 1, p. 57-74. https://doi.org/10.1146/annurev-food-030216-030216
Cheng, Y., Chen, S., Weng, C. 2006. Investigation of Goats Milk Adulteration with Cows Milk by PCR. Asian-Australasian Journal Of Animal Sciences, vol. 19, no. 10, p. 1503-1507. https://doi.org/10.5713/ajas.2006.1503
Di Domenico, M., Di Giuseppe, M., Wicochea Rodríguez, J., 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, vol. 100, no. 1, p. 106-112. https://doi.org/10.3168/jds.2016-11695
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. Journal Of Agricultural And Food Chemistry, vol. 60, no. 42, p. 10480-10485. https://doi.org/10.1021/jf3029896
Haenlein, G. 2004. Goat milk in human nutrition. Small Ruminant Research, vol. 51, no. 2, p. 155-163. https://doi.org/10.1016/j.smallrumres.2003.08.010
Hutu, I., Boldura, O., Popescu, S., Balta, C., Chis, C., Mircu, C. 2013. A multiplex PCR approach to the quantification of cow milk in goat products. Current Opinion in Biotechnology, vol. 24, no. 1, p. 95. https://doi.org/10.1016/j.copbio.2013.05.285
Jung, Y., Jhon, D., Kim, K., Hong, Y. 2011. Quantitative Detection of Cow Milk in Goat Milk Mixtures by Real-Time PCR. Korean Journal For Food Science Of Animal Resources, vol. 31, no. 6, p. 827-833. https://doi.org/10.5851/kosfa.2011.31.6.827
Klančnik, A., Toplak, N., Kovač, M., Ogrinc, N., Jeršek, B. 2015. Robust PCR-based method for quantification of bovine milk in cheeses made from caprine and ovine milk. International Journal of Dairy Technology, vol. 69, no. 4, p. 540-549. https://doi.org/10.1111/1471-0307.12287
Kemal Seçkin, A., Yilmaz, B., Tosun, H. 2017. Real-time PCR is a potential tool to determine the origin of milk used in cheese production. LWT - Food Science And Technology, vol. 77, p. 332-336. https://doi.org/10.1016/j.lwt.2016.11.065
Kotowich, M., Adamczyk, E., Bania, J. 2007. Application of a Duplex - PCR for detection of cow's milk in goat milk. Annals of Agricultural and Environmental Medicine, vol. 14, no. 2, p. 215-218.
Lanzilao, I., Burgalassi, F., Fancelli, S., Settimelli, M., Fani, R. 2005. Polymerase chain reaction-restriction fragment length polymorphism analysis of mitochondrial cyt-b gene from species of dairy interest. Journal of AOAC International, vol. 88, no. 1, p. 128-35. PMid:15759735
López-Calleja, I., González, I., Fajardo, V., Rodríguez, M., Hernández, P., García, T., Martín, R. 2004. Rapid Detection of Cows' Milk in Sheeps' and Goats' Milk by a Species-Specific Polymerase Chain Reaction Technique. Journal Of Dairy Science, vol. 87, no. 9, p. 2839-2845. https://doi.org/10.3168/jds.S0022-0302(04)73412-8
Mafra, I., Roxo, Á., Ferreira, I., Oliveira, M. 2007. A duplex polymerase chain reaction for the quantitative detection of cows' milk in goats' milk cheese. International Dairy Journal, vol. 17, no. 9, p. 1132-1138, https://doi.org/10.1016/j.idairyj.2007.01.009
Maudet, C., Taberlet, P. 2001. Detection of cows' milk in goats' cheeses inferred from mitochondrial DNA polymorphism. Journal Of Dairy Research, vol. 68, no. 2, p. 229-235. https://doi.org/10.1017/s0022029901004794
Maudet, C., Taberlet, P. 2002. Holstein's Milk Detection in Cheeses Inferred from Melanocortin Receptor 1 (MC1R) Gene Polymorphism. Journal of Dairy Science, vol. 85, no. 4, p. 707-715. https://doi.org/10.3168/jds.S0022-0302(02)74127-1
Mininni, A., Pellizzari, C., Cardazzo, B., Carraro, L., Balzan, S., Novelli, E. 2009. Evaluation of real-time PCR assays for detection and quantification of fraudulent addition of bovine milk to caprine and ovine milk for cheese manufacture. International Dairy Journal, vol. 19, no. 10, p. 617-623. https://doi.org/10.1016/j.idairyj.2009.04.003
Osman, A., Aradaib, I., Musa, O. 2013. Detection of Caprine-specific Nucleic Acid Sequences in Goat Milk Using Polymerase Chain Reaction. Materia Socio Medica, vol. 25, no. 2, p. 105. https://doi.org/10.5455/msm.2013.25.105-108
Rentsch, J., Weibel, S., Ruf, J., Eugster, A., Beck, K., Köppel, R. 2012. 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, vol. 236, no. 1, p. 217-227. https://doi.org/10.1007/s00217-012-1880-y
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. International Dairy Journal, vol. 28, no. 1, p. 32-35. https://doi.org/10.1016/j.idairyj.2012.08.006
Zeleňáková, L., Židek, R., Čanigová, M., Žiarovská, J., Zajác, P., Maršálková, L., Fikselová, M., Golian, J. 2016. Research And Practice: Quantification Of Raw And Heat-Treated Cow Milk in Sheep Milk, Cheese And Bryndza By ELISA Method. Potravinarstvo, vol. 10, no. 1, p. 14-22. https://doi.org/10.5219/566
Zhang, C., Fowler, M., Scott, N., 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, vol. 18, no. 9, p. 1149-1158. https://doi.org/10.1016/j.foodcont.2006.07.018
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