<<<  Back

2022, № 96

UDC: 575.113.2, 636.2.034
GSNTI: 34.15.05, 68.39.29

Development of a genetic test system: Design of fluorescent-labeled primers for real-time PCR and optimization of assay for a1/a2 milk conditions

Genetic variants of milk proteins have been of great interest for decades, as they are associated with important problems, such as the qualitative composition and technological properties of milk and dairy products. Relatively recently, the "hypothesis A1 / A2" was put forward. This hypothesis states that the β-casein (β-CN) A1 variant may be a dietary risk factor for cardiovascular disease, type 1 diabetes, sudden infant death syndrome, and neurological disorders due to the release of a specific polypeptide, β-casomorphin-7(BCM7 ) (60-66 amino acid residues of β-CN) is a peptide derived from beta-casein variants bearing His67 (type A1) instead of Pro67 (type A2). At the same time, no evidence of such side effects for natural peptides derived from A2 β-casein is expected or noted. Due to the importance of determining both the carriage of certain milk type-controlling single nucleotide polymorphisms in cows and the presence of BCM7 in milk and its products, a number of approaches have now been developed for the analysis of A1/A2 milk. The main approaches can be divided into two groups, according to the applied laboratory honey badgers. In one case, to directly determine the content of β-casomorphin-7 in milk and dairy products, methods of biochemical analysis are used, separating milk proteins using isoelectric focusing, determining the isoelectric points of the corresponding peptides. In another case, various modifications of the polymerase chain reaction (PCR) method are used for analysis. This work is the second part of the publication, which describes the design of specific fluorescently labeled primers for use in the original genetic test system for the analysis of A1/A2 milk developed by the authors. The approach is based on the use of a new polymerase - called SNPase (aka SNPdetect) DNA polymerase - today it is the best enzyme for conducting allele-specific PCR reactions. In the Russian Federation, the described system is the only one based on this principle, and has a number of serious advantages over the previously used methods for analyzing A1 / A2 milk. In addition, the proposed article describes the optimized conditions for the proposed genetic test system and provides examples of using the approach to analyze a representative sample of several dozen high-breed animals from the herd of the Krasnodarskoye experimental farm of the Kuban State Agrarian University.
Keywords: Mutation, polypeptide, hypersensitivity, A2-milk, restriction fragment length polymorphism (RFLP), real-time polymerase chain reaction, herd allelic frequencies.
DOI: 10.21515/1999-1703-96-247-258

References:

1. Bonfatti, V. Effects of β-κ-casein (CSN2-CSN3) haplotypes, β-lactoglobulin (BLG) genotypes, and detailed protein composition on coagulation properties of individual milk of Simmental cows / V. Bonfatti, G. Di Martino, A. Cecchinato, L. Degano & P. Carnier // Journal of Dairy Science. - 2010. - 93(8). - Р. 3809-3817. https://doi.org/10.3168/jds.2009-2779.
2. Caroli, A. M. Milk protein polymorphisms in cattle: Effect on animal breeding and human nutrition / A. M. Caroli, S. Chessa & G. J. Erhardt // Journal Dairy Science. - 2009. - 92(11). - 5335-5352. https://doi.org/10.3168/jds.2009-2461.
3. Farrell, H. M. Nomenclature of the proteins of cows’ milk - Sixth revision / H. M. Farrell, R. Jimenez-Flores, G. T. Bleck, E. M. Brown, J. E. Butler, L. K. Creamer et al. // Journal of Dairy Science. - 2004. - 87(6). - 1641-1674. https://doi.org/10.3168/jds.S0022-0302(04)73319-6.
4. Jakob, E. Technological properties of milk as influenced by genetic polymorphism of milk proteins - A review / E. Jakob & Z. Puhan (1992). - International Dairy Journal. - 2(3). - 157-178. https://doi.org/10.1016/0958-6946(92)90014-D.
5. Ketto, I. A. Effects of milk protein polymorphism and composition, casein micelle size and salt distribution on the milk coagulation properties in Norwegian Red cattle / I. A. Ketto, T. M. Knutsen, J. Øyaas, B. Heringstad, T. Ådnøy, T. G. Devold et al. // International Dairy Journal. - 2017. - 70. - 55-64. https://doi.org/10.1016/j.idairyj.2016.10.010.
6. Mayer, H. K.Composite milk protein phenotypes in relation to composition and cheesemaking properties of milk / H. K. Mayer, M. Ortner, E. Tschager & W. Ginzinger. -- International Dairy Journal. - 1997. - 7(5). - 305-310. https://doi.org/10.1016/S0958-6946(97)00019-8.
7. Lv, Z. Changes in metabolites from bovine milk with β-casein variants revealed by metabolomics / Z. Lv, H. Liu, Y. Yang, D. Bu, C. Zang, K. Yang // Animals. - 2020. - 10(6). - 954. https://doi.org/10.3390/ani10060954.
8. Ehrmann, S. Quantification of gene effects on single milk proteins in selected groups of dairy cows / S. Ehrmann, H. Bartenschlager & H. Geldermann // Journal of Animal Breeding and Genetics. - 1997. - 114(1-6). - 121-132. https://doi.org/10.1111/j.1439-0388.1997.tb00499.x.
9. Kaminski, ´S. Polymorphism of bovine beta-casein and its potential effect on human health / ´S Kaminski, ´A. Cie´slinska & E. Kostyra // Journal of Applied Genetics. - 2007. - 48(3). - 189-198. https://doi.org/10.1007/BF03195213
10. Godert, ¨ M. Background of beta-casein A2 in bovine milk - Strategies and limitations by breeding and the dairy industry with regard to a possible new demand / ¨ M. Godert, H. Brandt & G. Erhardt // Züchtungskunde. - 2017. - 89(6). - 451-474. https://www.zuechtungskunde.de/artikel.dll/zueku-2017-06-goedert-et-al_NjA5 MDQ2NA.PDF? UID =B62598BCA4C96C5A2A3705AE484E69331 CA588B4C9A052B1.
11. Mayer, H. K. Milk protein polymorphism in Austrian dairy cattle breeds / H. K. Mayer, A. Marchler, C. Prohaska & R. Norz (1997a) // Milchwissenschaft. - 52(7). - 366-369.
12. Farrell, H. M. Nomenclature of the proteins of cows’ milk - Sixth revision / H. M. Farrell, R. Jimenez-Flores, G. T. Bleck, E. M. Brown, J. E. Butler, L. K. Creamer et al. // Journal of Dairy Science. - 2004. - 87(6). - 1641-1674. https://doi.org/10.3168/jds.S0022-0302(04)73319-6.
13. Gallinat, J. L. DNA-based identification of novel bovine casein gene variants /j. L. Gallinat, S. Qanbari, ¨ C. Drogemüller, E. C. Pimentel, G. Thaller & J. Tetens // Journal of Dairy Science, - 2013. - 96(1). - 699-709. https://doi.org/10.3168/jds.2012-5908.
14. Cie´slinska,´ A. Milk from cows of different β-casein genotypes as a source of β-casomorphin7 / Cie´slinska,´ A., Kostyra, E., Kostyra, H., Olenski, ´ K., Fiedorowicz, E., & Kaminski, ´S. // International Journal of Food Sciences and Nutrition. - 2012. - 63(4). - 426-430. https://doi.org/10.3109/09637486.2011.634785.
15. Elliott, R. B. Type 1 (insulin-dependent) diabetes mellitus and cow milk: Casein variant consumption // R. B. Elliott, D. P. Harris, J. P. Hill, N. J. Bibby & H. E. Wasmuth // Diabetologia. -1999. - 42. - 292-296. https://doi.org/10.1007/s001250051153.
16. Ul Haq, M. R. Release of β-casomorphin-7/5 during simulated gastrointestinal digestion of milk β-casein variants from Indian crossbred cattle (Karan Fries) / M. R. Ul Haq, R. Kapila & S. Kapila // Food Chemistry. - 2015. - 168. - 70-79. https://doi.org/10.1016/j.foodchem.2014.07.024.
17. Truswell, A. S. The A2 milk case: A critical review / A. S. Truswell // European Journal of Clinical Nutrition. - 2005. - 59(5). - 623-631. https://doi.org/10.1038/sj.ejcn.1602104.
18. Küllenberg De Gaudry, D. Milk A1 β-casein and health-related outcomes in humans: A systematic review / D. Küllenberg De Gaudry, S. Lohner, C. Schmucker, P. Kapp, E. Motschall, ¨. S. Horrlein et al. // Nutrition Reviews. - 2019. - 77(5). - 278-306. https://doi.org/10.1093/nutrit/nuy063.
19. Oliveira Mendes, M. A2A2 milk: Brazilian consumers’ opinions and effect on sensory characteristics of Petit Suisse and Minas cheeses / M. Oliveira Mendes, M. Ferreira de Morais & J. Ferreira Rodrigues // LWT - Food Science and Technology. - 2019. - 108. - 207-213. https://doi.org/10.1016/j.lwt.2019.03.064.
20. Givens, I. Proportions of A1, A2, B and C β-casein protein variants in retail milk in the UK / I. Givens, P. Aikman, T. Gibson & R. Brown // Food Chemistry. - 2013. - 139(1-4). - 549-552. https://doi.org/10.1016/j.foodchem.2013.01.115.
21. Bentivoglio, D. Is there a promising market for the A2 milk? Analysis of Italian Consumer Preferences / D. Bentivoglio, A. Finco, G. Bucci & G. Staffolani // Sustainability. - 2020. - 12(17). - 6763. https://doi.org/10.3390/su12176763.
22. Massella, E. Evaluation of bovine beta casein polymorphism in two dairy farms located in northern Italy / E. Massella, S. Piva, F. Giacometti, G. Liuzzo, A. V. Zambrini & A. Serraino // Italian Journal of Food Safety. - 2017. - 6(3). - 131-133. https://doi.org/10.4081/ijfs.2017.6904.
23. De Noni, I. Release of β-casomorphins 5 and 7 during simulated gastro-intestinal digestion of bovine β-casein variants and milk-based infant formulas / I. De Noni // Food Chemistry. - 2008. - 110(4). - 897-903. https://doi.org/10.1016/j.foodchem.2008.02.077.
24. Hollar, C. M. Separation of β-casein A1, A2, and B using cation-exchange fast protein liquid chromatography / C. M. Hollar, A. J. R. Law, D. G. Dalgleish, J. F. Medrano & R. J. Brown // Journal of Dairy Science. - 1991. - 74(10). - 3308-3313. https://doi.org/10.3168/jds.S0022-0302(91)78517-2.
25. Bonfatti, V. Validation of a new reversed-phase high-performance liquid chromatography method for separation and quantification of bovine milk protein genetic variants // V. Bonfatti, L. Grigoletto, A. Cecchinato, L. Gallo & P. Carnier // Journal of Chromatography A. - 2008. - 1195(1-2). - 101-106. https://doi.org/10.1016/j.chroma.2008.04.075.
26. De Poi, R. Development of an LC-MS method for the identification of β-casein genetic variants in bovine milk / R. De Poi, E. De Dominicis, E. Gritti, F. Fiorese, S. Saner, & P. P. de Laureto // Food Analytical Methods. - 2020. - 13(12). - 2177-2187. https://doi.org/10.1007/s12161-020-01817.
27. Duarte-V´ azquez, M. A. Use of urea-polyacrylamide electrophoresis for discrimination of A1 and A2 beta casein variants in raw cow’s milk / M. A. Duarte-V´azquez, C. R. García-Ugalde, ´ B. E. Alvarez, L. M. Villegas, ´ B. E. García Almendarez, J. L. Rosado et al. // Journal of Food Science and Technology. - 2018. - 55(5). - 1942-1947. https://doi.org/10.1007/s13197-018-3088-z.
28. Braunschweig, M. H. Associations between casein haplotypes and milk traits of Braunvieh and Fleckvieh / M. H. Braunschweig (1998) // Doctoral thesis, ETH Zurich, Switzerland. - 1998). - DOI: 10.3929/ethz-a-001988576
29. Smiltina, D. Molecular genetics analysis of milk protein gene polymorphism of dairy cows and breeding bulls in Latvia / D. Smiltina & Z. Grislis // Agronomy Research. - 2018. - 16(3). - 900-909. https://doi.org/10.15159/AR.18.084.
30. Kuˇcerova, ´ J. Milk protein genes CSN1S1, CSN2, CSN3, LGB and their relation to genetic values of milk production parameters in Czech Fleckvieh / ´J. Kuˇcerova, A. Matˇejíˇcek, O. M. Jandurov´a, P. Sørensen, ´ E. Nˇemcova, M. Stípkov ˇ et al. // Czech Journal of Animal Science. - 2006. - 51(6). - 241-247. https://doi.org/10.17221/3935-cjas. ACRS.
31. Jann, O. A new variant in exon VII of bovine β-casein gene (CSN2) and its distribution among European cattle breeds // O. Jann, G. Ceriotti, A. Caroli, & G. Erhardt // Journal of Animal Breeding and Genetics. - 2002. - 119(1). - 65-68. https://doi.org/10.1046/j.1439-0388.2002.00318.x.
32. Chessa, S. Development of a SNP genotyping microarray platform for the identification of bovine milk protein genetic polymorphisms / S. Chessa, F. Chiatti, G. Ceriotti, A. Caroli, C. Consolandi, G. Pagnacco et al. // Journal of Dairy Science. - 2007. - 90(1). - 451-464. https://doi.org/10.3168/jds.S0022-0302(07)72647-4
33. Giglioti, R. New high-sensitive rhAmp method for A1 allele detection in A2 milk samples / R. Giglioti, G. Gutmanis, L. M. Katiki, C. H. Okino, M. C. de Sena Oliveira & A. E. Vercesi Filho // Food Chemistry. - 2020. - 313(5). - Article 126167. https://doi.org/10.1016/j. foodchem.2020.126167.
34. https://evrogen.ru/kit-user-manuals/SNP detect.pdf; (Официальный сайт ООО «Евроген», Москва, Россия; время обращения 11.05.2022)
35. Stadler, J. SNPaseARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients /j. Stadler, J. Eder, B. Pratscher, S. Brandt, D. Schneller, R. Müllegger et al. // PLoS ONE 10(11): e0142273. - 2015. DOI: 10.1371/ journal.pone.0142273.
36. Князева, В. В. Разработка генетической тестовой системы: Получение фрагментов гена β-казеина крупного рогатого скота, содержащих нуклеотидные замены, характерные для различных аллельных форм белка / В. В. Князева, А. В. Гарковенко, В. В. Радченко, А. Г. Кощаев // Труды Кубанского государственного аграрного университета. - 2022. - № 2(95). - С. 157-164.
37. Гладырь, Е. А. Молекулярная диагностика генетического дефекта FMO в айрширской породе крупного рогатого скота / Е. А. Гладырь, Е. Н. Коновалова, О. В. Костюнина, А. Г. Кощаев // Труды Кубанского государственного аграрного университета. - 2019. - № 80. - С. 215-221.
38. Кощаев, А. Г. Генетическое разнообразие крупного рогатого скота, разводимого в Краснодарском крае / А. Г. Кощаев, С. Ю. Шуклин, И. В. Щукина // Аграрный вестник Урала. - 2017. - № 12 (166). - С. 5.

Authors:

1. Knyazeva Valeriya Vladimirovna, graduate student, Federal State Budgetary Educational Institution of Higher Education “I.T. Trubilin Kuban State Agrarian University”.
2. Garkovenko Alexey Vyacheslavovich, master of biological sciences, CEOS, LLC “DNA Expertise”; Federal State Budgetary Educational Institution of Higher Education “I.T. Trubilin Kuban State Agrarian University”.
3. Radchenko Vitaliy Vladislavovich, PhD in Biology, leading researcher, LLC “DNA Expertise”.
4. Koshchaev Andrei Georgievich, DSc in Biology, professor, Federal State Budgetary Educational Institution of Higher Education “I.T. Trubilin Kuban tate Agrarian University”.