Detecting soybean and milk in dairy and soy products with post-PCR high resolution melting assays

Sovová T., Křížová B., Kučera L., Ovesná J. (2020): Detecting soybean and milk in dairy and soy products with post-PCR high resolution melting assays. Czech J. Food Sci., 38: 209–214.

download PDF

We have developed and validated post-PCR (polymerase chain reaction) high resolution melting (HRM) based assays that allow identifying the presence of two major allergenic foods – soybean and bovine milk – in food products. A new set of primers for PCR was developed for detection of the gene encoding the soybean protein lectin. The assay was validated using reference samples and used for the analysis of artificially prepared mixed samples of dairy and soy products of different matrices, as well as of real products available in the market (spray creams). The limits of detection (LODs) of the soybean (8 copies) and bovine milk (4 copies) assays were lower compared to LODs of other previously published PCR-based assays. The analysis of several commercial spray creams revealed an undeclared presence of soybean in one of the samples. The newly developed HRM assays are precise and robust alternatives for the control of food composition and falsification by competent authorities.

Berdal K.G., Holst-Jensen A. (2001): Roundup Ready® soybean event-specific real-time quantitative PCR assay and estimation of the practical detection and quantification limits in GMO analyses. European Food Research and Technology, 213: 432–438.
Costa J., Amaral J.S., Grazina L., Oliveira M.B.P.P., Mafra I. (2017): Matrix-normalised real-time PCR approach to quantify soybean as a potential food allergen as affected by thermal processing. Food Chemistry, 221: 1843–1850.
Dalmasso A., Civera T., La Neve F., Bottero M.T. (2011): Simultaneous detection of cow and buffalo milk in mozzarella cheese by real-time PCR assay. Food Chemistry, 124: 362–366.
Espiñeira M., Herrero B., Vieites J.M., Santaclara F.J. (2010): Validation of end-point and real-time PCR methods for the rapid detection of soy allergen in processed products. Food Additives & Contaminants: Part A, 27: 426–432.
Feligini M., Bonizzi I., Cubric Curik V., Parma P., Greppi G.F., Enne G. (2005): Detection of adulteration in Italian mozzarella cheese using mitochondrial DNA templates as biomarkers. Food Technology and Biotechnology, 43: 91–95.
Foucard T., Malmheden Yman I. (1999): A study on severe food reactions in Sweden – is soy protein an underestimated cause of food anaphylaxis? Allergy, 54: 261–265.
Ibáñez C., Acunha T., Valdés A., García-Cañas V., Cifuentes A., Simó C. (2016): Capillary Electrophoresis in Food and Foodomics. In: Schmitt-Kopplin P. (eds): Capillary Electrophoresis: Methods and Protocols. Springer New York, New York, NY, 1483: 471–507.
Jablonski J.E., Moore J.C., Harnly J.M. (2014): Nontargeted detection of adulteration of skim milk powder with foreign proteins using UHPLC–UV. Journal of Agricultural and Food Chemistry, 62: 5198–5206.
Lockley A.K., Bardsley R.G. (2000): DNA-based methods for food authentication. Trends in Food Science & Technology, 11: 67–77.
López-Calleja I., Alonso I.G., Fajardo V., Rodríguez M.A., Hernández P.E., García T., Martín R. (2005): PCR detection of cows’ milk in water buffalo milk and mozzarella cheese. International Dairy Journal, 15: 1122–1129.
López-Calleja I., González I., Fajardo V., Martín I., Hernández P.E., García T., Martín R. (2007): Real-time TaqMan PCR for quantitative detection of cows’ milk in ewes’ milk mixtures. International Dairy Journal, 17: 729–736.
Mafra I., Ferreira I.M.P.L.V.O., Faria M.A., Oliveira B.P.P. (2004): A novel approach to the quantification of bovine milk in ovine cheeses using a duplex polymerase chain reaction method. Journal of Agricultural and Food Chemistry, 52: 4943–4947.
Mayer H.K., Bürger J., Kaar N. (2012): Quantification of cow’s milk percentage in dairy products – a myth? Analytical and Bioanalytical Chemistry, 403: 3031–3040.
Nwaru B.I., Hickstein L., Panesar S.S., Roberts G., Muraro A., Sheikh A. (2014): Prevalence of common food allergies in Europe: A systematic review and meta-analysis. Allergy, 69: 992–1007.
Phipps R.H., Beever D.E., Humphries D.J. (2002): Detection of transgenic DNA in milk from cows receiving herbicide tolerant (CP4EPSPS) soyabean meal. Livestock Production Science, 74: 269–273.
Phipps R.H., Deaville E.R., Maddison B.C. (2003): Detection of transgenic and endogenous plant DNA in rumen fluid, duodenal digesta, milk, blood, and feces of lactating dairy cows. Journal of Dairy Science, 86: 4070–4078.
Rani A., Sharma V., Arora S., Lal D., Kumar A. (2015): A rapid reversed–phase thin layer chromatographic protocol for detection of adulteration in ghee (clarified milk fat) with vegetable oils. Journal of Food Science and Technology, 52: 2434–2439.
Rea S., Chikuni K., Branciari R., Sangamayya R.S., 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. Journal of Dairy Research, 68: 689–698.
Ren Q.R., Zhang H., Guo H.Y., Jiang L., Tian M., Ren F.Z. (2014): Detection of cow milk adulteration in yak milk by ELISA. Journal of Dairy Science, 97: 6000–6006.
Rott M.E., Lawrence T.S., Wall E.M., Green M.J. (2004): Detection and quantification of roundup ready soy in foods by conventional and real-time polymerase chain reaction. Journal of Agricultural and Food Chemistry, 52: 5223–5232.
Sakaridis I., Ganopoulos I., Argiriou A., Tsaftaris A. (2013a): A fast and accurate method for controlling the correct labeling of products containing buffalo meat using High Resolution Melting (HRM) analysis. Meat Science, 94: 84–88.
Sakaridis I., Ganopoulos I., Argiriou A., Tsaftaris A. (2013b): High resolution melting analysis for quantitative detection of bovine milk in pure water buffalo mozzarella and other buffalo dairy products. International Dairy Journal, 28: 32–35.
Song H., Xue H., Han Y. (2011): Detection of cow’s milk in Shaanxi goat’s milk with an ELISA assay. Food Control, 22: 883–887.
Walker J.A., Hughes D.A., Anders B.A., Shewale J., Sinha S.K., Batzer M.A. (2003): Quantitative intra-short interspersed element PCR for species-specific DNA identification. Analytical Biochemistry, 316: 259–269.
Wu R., Wang Z., Fung Y.S., Peng Seah D.Y., Yeung W.S.B. (2014): Assessment of adulteration of soybean proteins in dairy products by 2D microchip-CE device. Electrophoresis, 35: 1728–1734.
download PDF

© 2021 Czech Academy of Agricultural Sciences | Prohlášení o přístupnosti