meatTheory and practice of meat processingТеория и практика переработки мяса2414-438X2414-441XФГБНУ «Федеральный научный центр пищевых систем им. В.М. Горбатова» РАН10.21323/2414-438X-2020-5-3-28-31meat-145Research ArticleСтатьиDevelopment of high sensitive real-time PCR to detect mustard and other allergens of the family Brassicaceae in food sampleshttps://orcid.org/0000-0003-1348-860XKurbakovK. A.

Konstantin A. Kurbakov — Engineer of Laboratory of hygiene of manufacture and microbiology

109316, Moscow, Talalikhina str., 26. Tel: +7–495–676–63–21

homo_ludens@vniimp.ruhttps://orcid.org/0000-0001-9063-2164ZhulinkovaV. N.

Valentina N. Zhulinkova — Engineer of Laboratory of hygiene of manufacture and microbiology

109316, Moscow, Talalikhina str., 26. Tel: +7–495–676–63–21

v.zhulinkova@fncpc.ruhttps://orcid.org/0000-0002-0038-9744MinaevM. Yu.

Mihail Yu. Minaev — candidate of technical sciences, head of Laboratory of molecular biology and bioinformatics

109316, Moscow, Talalikhina str., 26. Tel.: +7–495–676–79–81

mminaev@inbox.ruV. M. Gorbatov Federal Research Center for Food Systems of Russian Academy of SciencesRussian Federation202026102020532831Copyright © Kurbakov K.A., Zhulinkova V.N., Minaev M.Y., 20202020Kurbakov K.A., Zhulinkova V.N., Minaev M.Y.Kurbakov K.A., Zhulinkova V.N., Minaev M.Y.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.meatjournal.ru/jour/article/view/145

Mustard is a commonly used condiment including in production of other food products. As mustard is an allergen, it is necessary to control its presence. The development of PCR test-systems for its detection is complicated by the fact that this condiment can be made from seeds of various plant species (Brassica juncea, Brassica nigra, Sinapis alba) of the family Brassicaceae that are not closely related. This family includes other plant species such as white cabbage (Brassica oleracea) and rapeseed (Brassica napus), which can cause the allergic reaction, although seldom. In this connection, many authors use primers specific to many species of this family, including to allergens, to detect mustard. In this work, we used the similar strategy. To increase sensitivity, primers for the mitochondrial COX gene were selected. To increase PCR stability in analysis of deeply processed products, primers were selected for a region with a length of 61 base pair. In the work, the specificity and sensitivity of the developed PCR method was confirmed. Analyses of different products, including those that underwent deep technological processing, were carried out with these primers. Also, primers were selected to detect white mustard (S. alba). When analyzing products on the presence of white mustard, charac‑ teristic regional preferences were demonstrated: this species is used in manufacturing products mainly in the UK and USA.

real-time PCRBrassica junceaBrassica nigraSinapis albaBrassicaceaeallergensfood productsReferencesTechnical regulations of the Customs Union TR CU022/2011 “Food products in terms of its labeling” (approved by the decision of the customs Union Commission of December 9, 2011 № 880). Moscow, — 2011. (in Russian)Technical regulations of the Customs Union TR CU022/2011 “Food products in terms of its labeling” (approved by the decision of the customs Union Commission of December 9, 2011 № 880). Moscow, — 2011. (in Russian)Regulation (EU) No 1169/2011 of 25 October 2011 on the provision of food information to consumers, amending Regulations (EC) No. 1924/2006 and (EC) No. 1925/2006 of the European Parliament and of the Council, and repealing Commission Directive 87/250/EEC, Council Directive 90/496/EEC, Commission Directive 1999/10/EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and 2008/5/EC and Commission Regulation (EC) No 608/2004. Official Journal of the European Union, L304, 18–63.Regulation (EU) No 1169/2011 of 25 October 2011 on the provision of food information to consumers, amending Regulations (EC) No. 1924/2006 and (EC) No. 1925/2006 of the European Parliament and of the Council, and repealing Commission Directive 87/250/EEC, Council Directive 90/496/EEC, Commission Directive 1999/10/EC, Directive 2000/13/EC of the European Parliament and of the Council, Commission Directives 2002/67/EC and 2008/5/EC and Commission Regulation (EC) No 608/2004. Official Journal of the European Union, L304, 18–63.Figueroa, J., Blanco, C., Dumpiérrez, A.G., Almeida, L., Ortega, N., Castillo, R., Navarro, L., Pérez, E., Gallego, M.D., Carrillo, T. (2005). Mustard allergy confirmed by double-blind placebo-controlled food challenges: clinical features and cross-reactivity with mugwort pollen and plant-derived foods. Allergy, 60(1), 48–55. https://doi.org/10.1111/j.1398–9995.2005.00644.xFigueroa, J., Blanco, C., Dumpiérrez, A.G., Almeida, L., Ortega, N., Castillo, R., Navarro, L., Pérez, E., Gallego, M.D., Carrillo, T. (2005). Mustard allergy confirmed by double-blind placebo-controlled food challenges: clinical features and cross-reactivity with mugwort pollen and plant-derived foods. Allergy, 60(1), 48–55. https://doi.org/10.1111/j.1398–9995.2005.00644.xMenéndes-Arias, L., Moneo, I., Domínguez, J., Rodrígues, R. (1988). Primary structure of the major allergen of yellow mustard (Sinapis alba L.) seed, Sin a I. European Journal of Biochemistry, 177(1), 159–166. https://doi.org/10.1111/j.1432–1033.1988.tb14357.x-i2Menéndes-Arias, L., Moneo, I., Domínguez, J., Rodrígues, R. (1988). Primary structure of the major allergen of yellow mustard (Sinapis alba L.) seed, Sin a I. European Journal of Biochemistry, 177(1), 159–166. https://doi.org/10.1111/j.1432–1033.1988.tb14357.x-i2de la Peña, M. A. G., Menéndez-Arias, L., Monsalve, R. I., Rodríguez, R. (1991). Isolation and Characterization of a Major Allergen from Oriental Mustard Seeds, Bra j1. International Archives of Allergy and Immunology, 96(3), 263–270. https://doi.org/10.1159/000235505de la Peña, M. A. G., Menéndez-Arias, L., Monsalve, R. I., Rodríguez, R. (1991). Isolation and Characterization of a Major Allergen from Oriental Mustard Seeds, Bra j1. International Archives of Allergy and Immunology, 96(3), 263–270. https://doi.org/10.1159/000235505Palomares, O., Cuesta-Herranz, J., Vereda, A., Sirvent, S., Villalba, M., Rodríguez, R. (2005). Isolation and identification of an 11S globulin as a new major allergen in mustard seeds. Annals of Allergy, Asthma & Immunology, 94(5), 586–592. https://doi.org/10.1016/S1081–1206(10)61138–6Palomares, O., Cuesta-Herranz, J., Vereda, A., Sirvent, S., Villalba, M., Rodríguez, R. (2005). Isolation and identification of an 11S globulin as a new major allergen in mustard seeds. Annals of Allergy, Asthma & Immunology, 94(5), 586–592. https://doi.org/10.1016/S1081–1206(10)61138–6Angelina, A., Sirvent, S., Palladino, C., Vereda, A., Cuesta- Herranz, J., Eiwegger, T., Rodríguez, R., Breiteneder, H., Villalba, M., Palomares, O. (2016). The lipid interaction capacity of Sin a 2 and Ara h 1, major mustard and peanut allergens of the cupin superfamily, endorses allergenicity. Allergy, 71(9), 1284–1294. https://doi.org/10.1111/all.12887Angelina, A., Sirvent, S., Palladino, C., Vereda, A., Cuesta- Herranz, J., Eiwegger, T., Rodríguez, R., Breiteneder, H., Villalba, M., Palomares, O. (2016). The lipid interaction capacity of Sin a 2 and Ara h 1, major mustard and peanut allergens of the cupin superfamily, endorses allergenicity. Allergy, 71(9), 1284–1294. https://doi.org/10.1111/all.12887Hermanides, H. K., Laheÿ-de Boer, A. M., Zuidmeer, L., Gui‑ kers, C., Ree, R., Knulst, A. C. (2006). Brassica oleracea pollen, a new source of occupational allergens. Allergy, 61(4), 498–502. https://doi.org/10.1111/j.1398–9995.2006.01055.xHermanides, H. K., Laheÿ-de Boer, A. M., Zuidmeer, L., Gui‑ kers, C., Ree, R., Knulst, A. C. (2006). Brassica oleracea pollen, a new source of occupational allergens. Allergy, 61(4), 498–502. https://doi.org/10.1111/j.1398–9995.2006.01055.xKhaliq, B., Falke, S., Negm, A., Buck, F., Munawar, A., Saqib, M., Mahmood, S., Ahmad, M.S., Betzel, C., Akrem, A. (2017). SAXS and other spectroscopic analysis of 12S cruciferin isolated from the seeds of Brassica nigra. Journal of Molecular Structure, 1137, 60–66. https://doi.org/10.1016/j.molstruc.2017.02.043Khaliq, B., Falke, S., Negm, A., Buck, F., Munawar, A., Saqib, M., Mahmood, S., Ahmad, M.S., Betzel, C., Akrem, A. (2017). SAXS and other spectroscopic analysis of 12S cruciferin isolated from the seeds of Brassica nigra. Journal of Molecular Structure, 1137, 60–66. https://doi.org/10.1016/j.molstruc.2017.02.043Lee, P. W., Hefle, S. L., Taylor, S. L. Lee, P.-W., Hefle, S.L., Taylor, S.L. (2008). Sandwich enzyme-linked immunosorbent assay (ELISA) for detection of mustard in foods. Journal of food science, 73(4), T62-T68. https://doi.org/10.1111/j.1750–3841.2008.00725.xLee, P. W., Hefle, S. L., Taylor, S. L. Lee, P.-W., Hefle, S.L., Taylor, S.L. (2008). Sandwich enzyme-linked immunosorbent assay (ELISA) for detection of mustard in foods. Journal of food science, 73(4), T62-T68. https://doi.org/10.1111/j.1750–3841.2008.00725.xKoppelman, S. J., Vlooswijk, R., Bottger, G., Van Duijn, G., Van der Schaft, P, Dekker, J., Van Bergen, H. (2007). Development of an enzyme-linked immunosorbent assay method to detect mustard protein in mustard seed oil. Journal of food protection, 70(1), 179–183. https://doi.org/10.4315/0362–028x‑70.1.179Koppelman, S. J., Vlooswijk, R., Bottger, G., Van Duijn, G., Van der Schaft, P, Dekker, J., Van Bergen, H. (2007). Development of an enzyme-linked immunosorbent assay method to detect mustard protein in mustard seed oil. Journal of food protection, 70(1), 179–183. https://doi.org/10.4315/0362–028x‑70.1.179Palle-Reisch, M., Wolny, M., Cichna-Markl, M., Hochegger, R. (2013). Development and validation of a real-time PCR method for the simultaneous detection of black mustard (Brassica nigra) and brown mustard (Brassica juncea) in food. Food chemistry, 138(1), 348–355. https://doi.org/10.1016/j.foodchem.2012.10.055Palle-Reisch, M., Wolny, M., Cichna-Markl, M., Hochegger, R. (2013). Development and validation of a real-time PCR method for the simultaneous detection of black mustard (Brassica nigra) and brown mustard (Brassica juncea) in food. Food chemistry, 138(1), 348–355. https://doi.org/10.1016/j.foodchem.2012.10.055Palle-Reisch, M., Cichna-Markl, M., Hochegger, R. (2014). Development and validation of a duplex real-time PCR assay for the simultaneous detection of three mustard species (Sinapis alba, Brassica nigra and Brassica juncea) in food. Food chemistry, 153, 66–73. https://doi.org/10.1016/j.foodchem.2013.12.035Palle-Reisch, M., Cichna-Markl, M., Hochegger, R. (2014). Development and validation of a duplex real-time PCR assay for the simultaneous detection of three mustard species (Sinapis alba, Brassica nigra and Brassica juncea) in food. Food chemistry, 153, 66–73. https://doi.org/10.1016/j.foodchem.2013.12.035Köppel, R., van Velsen-Zimmerli, F., Bucher, T. (2012). Two quantitative hexaplex real-time PCR systems for the detection and quantification of DNA from twelve allergens in food. European Food Research and Technology, 235(5), 843–852. https://doi.org/10.1007/s00217–012–1806–8Köppel, R., van Velsen-Zimmerli, F., Bucher, T. (2012). Two quantitative hexaplex real-time PCR systems for the detection and quantification of DNA from twelve allergens in food. European Food Research and Technology, 235(5), 843–852. https://doi.org/10.1007/s00217–012–1806–8Mano, J., Nishitsuji, Y., Kikuchi, Y., Fukudome, S., Hayashida, T., Kawakami, H., Kurimoto, Y., Noguchi, A., Kondo, K., Teshima, R., Takabatake, R., Kitta, K.(2017). Quantification of DNA fragmenta‑ tion in processed foods using real-time PCR. Food chemistry, 226, 149–155. https://doi.org/10.1016/j.foodchem.2017.01.064Mano, J., Nishitsuji, Y., Kikuchi, Y., Fukudome, S., Hayashida, T., Kawakami, H., Kurimoto, Y., Noguchi, A., Kondo, K., Teshima, R., Takabatake, R., Kitta, K.(2017). Quantification of DNA fragmenta‑ tion in processed foods using real-time PCR. Food chemistry, 226, 149–155. https://doi.org/10.1016/j.foodchem.2017.01.064GenBank®. Bethesda, MD, USA: National Center for Biotech‑ nology Information (NCBI), US National Library of Medicine; 2017. [Electronic resource: http://www.ncbi.nlm.nih.gov/ Access date 14.07.2020]GenBank®. Bethesda, MD, USA: National Center for Biotech‑ nology Information (NCBI), US National Library of Medicine; 2017. [Electronic resource: http://www.ncbi.nlm.nih.gov/ Access date 14.07.2020]Primer-BLAST. Bethesda, MD, USA: National Center for Bio‑ technology Information, U. S. National Library of Medicine; 2017. [Electronic resource: https://www.ncbi.nlm.nih.gov/tools/prim‑ er-blast/index.cgi Access date 14.07.2020]Primer-BLAST. Bethesda, MD, USA: National Center for Bio‑ technology Information, U. S. National Library of Medicine; 2017. [Electronic resource: https://www.ncbi.nlm.nih.gov/tools/prim‑ er-blast/index.cgi Access date 14.07.2020]OligoAnalyzer 3.1, Integrated DNA Technologies, Inc., Coralville, IA, USA; 2017. [Electronic resource: http://eu.idtdna.com/calc/analyzer Access date 14.07.2020]OligoAnalyzer 3.1, Integrated DNA Technologies, Inc., Coralville, IA, USA; 2017. [Electronic resource: http://eu.idtdna.com/calc/analyzer Access date 14.07.2020]Microsoft Excel 2010, Microsoft, Redmond, WA, USA; 2016.Microsoft Excel 2010, Microsoft, Redmond, WA, USA; 2016.Bauer, T., Hammes, W. P., Haase, N. U., Hertel, C. (2004). Effect of food components and processing parameters on DNA degradation in food. Environmental Biosafety Research, 3(4), 215–223. https://doi.org/10.1051/ebr:2005005.Bauer, T., Hammes, W. P., Haase, N. U., Hertel, C. (2004). Effect of food components and processing parameters on DNA degradation in food. Environmental Biosafety Research, 3(4), 215–223. https://doi.org/10.1051/ebr:2005005.

The authors declare that there are no conflicts of interest present.