Detection of Lupine (Lupinus spp. L.) as a food allergen using three methods: end-point PCR, Real-Time PCR and Elisa

Authors

  • Ondrej Revák Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Food Hygiene and Safety,Tr. Andreja Hlinku 2, 949 76 Nitra
  • Jozef Golian Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Food Hygiene and Safety, Tr. A. Hlinku 2, 949 76 Nitra
  • Radoslav Židek Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Food Hygiene and Safety, Tr. A. Hlinku 2, 949 76 Nitra
  • Jozef Čapla Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Food Hygiene and Safety, Tr. A. Hlinku 2, 949 76 Nitra
  • Peter Zajác Slovak University of Agriculture in Nitra, Faculty of Biotechnology and Food Sciences, Department of Food Hygiene and Safety, Tr. A. Hlinku 2, 949 76 Nitra

DOI:

https://doi.org/10.5219/384

Keywords:

lupine, food allergy, end-point PCR, real-time PCR, ELISA

Abstract

The aim of this work was to compare three methods for the detection and quantification of lupine as an allergen in food. The methods that were used in this work were the direct method: ELISA and the indirect methods: end-point PCR and real-time PCR. We examined the detection limit (the sensitivity with which we can detect the presence of the allergen in a sample) and the reliability for performing an analysis. We used 17 samples of plant species from a processing plant for dehydrated soups production and lupine samples from lupine processing companies. Its practical use is wide and it is used mainly in the bakery industry, in the manufacture of confectionery, pasta, sauces, as a substitute for soy and also in the production of gluten-free food, because it does not contain gluten. Lupine, however, is also included in the list of 14 allergenic substances, which in accordance with the EU legislation must be listed on food labels. The high risk group, which suffers from primary sensitization or cross-reaction with peanuts, are allergic patients. In the EU, people who are allergic to peanuts range from 0.7 to 1.5%. In experiment 1, we detected the presence of lupine using primers for the detection of α- and δ-conglutine in the samples, using the end-point PCR method and the detection limit of this reaction was at the level of 100 ppm. For the vizualization of the DNA fragments, we used a 2% agarose gel and UV visualizer. In experiment 2 we detected lupine using the TaqMan real-time PCR reaction and primers for the detection of α and δ-conglutine at the level of 10 ppm of lupine in sample. The CP values of lupine using primers for the detection of α-conglutine was 24.85 ± 0.12 and the reliability equation was R2 = 0.9767. The CP lupine values using primers for the detection of δ-conglutine was 22.52 ± 0.17 and the reliability equation was R2 = 0.9925. In experiment 3, we used a sandwich ELISA method for the detection of lupine and the detection limit was within the range of 2-30 ppm and the reliability of the method according to the reliability equation was R2 = 0.9975. The high sensitivity and equation of reliability justify the use of these methods for the detection and quantification of lupine in practice. The most sensitive indirect method for the detection of lupine in our study was the method of real-time TaqMan PCR with a detection limit  10 000-10 ppm of lupine. The most sensitive direct methot was ELISA with detection limit 2-30 ppm of lupine.

Downloads

Download data is not yet available.

Metrics

Metrics Loading ...

References

Ballabio, C., Peñas, E., Uberti, F., Fiocchi, A., Duranti, M., Magni, C., Restani, P. 2013. Characterization of the sensitization profile to lupin in peanut-allergic children and assessment of cross-reactivity risk. Pediatr Allergy Immunol, vol. 24, no.3, p. 270-275. PMID: 23551124 DOI: https://doi.org/10.1111/pai.12054

Demmel, A. 2013. DNA-based Analysis of Food Allergens: Development and Validation of a Real-time PCR Method for the Detection of DNA from Lupine in Foods. Dissertation thessis, München: Technische Universität München, 115 p.

Demmel, A., Hupfer, A., Busch, U., Engel, K-H. 2011. Detection of lupine (Lupinus spp.) DNA in processed foods using real-time PCR. Food Control, vol. 22, no. 2, p. 215-220. https://doi.org/10.1016/j.foodcont.2010.07.001 DOI: https://doi.org/10.1016/j.foodcont.2010.07.001

Demmel, A., Hupfer, A., Busch, U., Engel, K-H. 2012. Quantification of lupine (Lupinus angustifolius) in wheat flour using real-time PCR and an internal standard material. Eur. Food Res. Technol., vol. 235, no. 1, p. 61-66. https://doi.org/10.1007/s00217-012-1741-8 DOI: https://doi.org/10.1007/s00217-012-1741-8

Dooper, M. M., Plassen, C., Holden, L., Lindvik, H., Faeste, C. K. 2009. Immunoglobulin E Cross-Reactivity Between Lupine Conglutins and Peanut Allergens in Serum of Lupine-Allergic Individuals. J. Investig. Allergol. Clin. Immunol., vol. 19, no. 4, p. 283-291. PMID: 19639724

Ecker, Ch., Ertl, A., Cichna-Markl, M. 2013. Development and Validation of Two Competitive ELISAs for the Detection of Potentially Allergenic Lupine (Lupinus Species) in Food. Food Analytical Methods, vol. 6, no. 1, p. 248-257. https://doi.org/10.1007/s12161-012-9418-2 DOI: https://doi.org/10.1007/s12161-012-9418-2

Edwards, A. C., van Barneveld, R. J. 1998. Lupins for Livestock and Fish, Lupins as Crop Plants: Biology, Production and Utilization. London : CAB International, 1998. p. 385-411. ISBN 978-0851992242.

Erbas, M., Certel, M., Uslu, M. K. 2005. Some chemical properties of white lupin seeds (Lupinus albus L.) Food Chemistry, vol. 89, no. 3, p. 341-345. https://doi.org/10.1016/j.foodchem.2004.02.040 DOI: https://doi.org/10.1016/j.foodchem.2004.02.040

Gomez-Galan, A. M. et al. 2010. Development of real-time PCR assays for the detection of lupin residues in food products. Eur. Food Res. Technol., vol. 230, no. 4, p. 597-608. https://doi.org/10.1007/s00217-009-1199-5 DOI: https://doi.org/10.1007/s00217-009-1199-5

Gomez-Galan, A. M., Brohée, M., Scaravelli, E., van Hengel, A. J., Chassaigne, H. 2011. Development of a real-time PCR method for the simultaneous detection of soya and lupin mitochondrial DNA as markers for the presence of allergens in processed food. Food Chemistry,

vol. 127, no. 2, p. 834-841. https://doi.org/10.1016/j.foodchem.2011.01.019 PMID: 23140743 DOI: https://doi.org/10.1016/j.foodchem.2011.01.019

Herridge, D. F., Doyle, A. D. 1988. The narrow-leafed lupin (Lupinus angustifolius L.) as a nitrogen-fixing rotation crop for cereal production. II. Estimates of fixation by field-grown crops. Australian Journal of Agricultural Research vol. 39, p. 1017-1028. https://doi.org/10.1071/AR9881017 DOI: https://doi.org/10.1071/AR9881017

Holden, L., Faeste, C. K., Egaas, E., 2005. Quantitative sandwich ELISA for the determination of lupine Lupinus spp. in food. Journal of Agricultural and Food Chemistry, vol. 53, no. 15, p. 5866-5871 https://doi.org/10.1021/jf050631i PMID: 16028967 DOI: https://doi.org/10.1021/jf050631i

Köppel, R., Dvorak, V., Zimmerli, F., Breitenmoser, A., Eugster, A., Waiblinger, H. U. 2010. Two tetraplex real-time PCR for the detection and quantification of DNA from eight allergens in food. Eur. Food Res. Technol., vol. 230, no. 3, p. 367-374. https://doi.org/10.1007/s00217-009-1164-3 DOI: https://doi.org/10.1007/s00217-009-1164-3

Mairal, T., Nadal, P., Svobodova, M., O'Sullivan, C. K. 2014. FRET-based dimeric aptamer probe for selective and sensitive Lup an 1 allergen detection, Biosensors and Bioelectronics, vol. 54, p. 207-210. https://doi.org/10.1016/j.bios.2013.10.070 PMID: 24280051 DOI: https://doi.org/10.1016/j.bios.2013.10.070

Martínez-Villaluenga, C., Frías, J., Vidal-Valverde, C. 2006. Functional lupin seeds (Lupinus albus L. and Lupinus luteus L.) after extraction of α-galactosides. Food Chemistry, vol. 98, no. 2 2006. p. 291-299. http//dx.doi/10.1016/j.foodchem.2005.05.074 DOI: https://doi.org/10.1016/j.foodchem.2005.05.074

Melo, T. S., Ferreira, R. B., Teixeira, A. N. 1994. The seed storage proteins from Lupinus albus. Phytochemistry, vol. 37, no. 3, p. 641-648. https://doi.org/10.1016/S0031-9422(00)90331-5 DOI: https://doi.org/10.1016/S0031-9422(00)90331-5

Peeters, K. A. B. M., Koppelman, S. J., Penninks, A. H., Lebens, A., Bruijnzeel-Koomen, C. A. F. M., Hefle, S. L., Taylor, S. L., Van Hoffen, E., Knulst, A. C. 2009. Clinical relevance of sensitization to lupine in peanut-sensitized adults. Allergy, vol. 64, no. 4, p. 549-555. https://doi.org/10.1111/j.1398-9995.2008.01818.x PMID: 19076544 DOI: https://doi.org/10.1111/j.1398-9995.2008.01818.x

Petterson, D. S., Harris, D. J. 1995. Cadmium and lead content of lupin seed grown in Western Australia. Australian Journal of Experimental Agriculture, vol. 35, no. 3, p. 403-407. https://doi.org/10.1071/EA9950403 DOI: https://doi.org/10.1071/EA9950403

Scarafoni, A., Ronchi, A., Duranti, M. 2009. A real-time PCR method for the detection and quantification of lupin flour in wheat flour-based matrices. Food Chemistry, vol. 115, no. 3, p. 1088-1093. https://doi.org/10.1016/j.foodchem.2008.12.087 DOI: https://doi.org/10.1016/j.foodchem.2008.12.087

Waiblinger, H. U., Boernsen, B., Näumann, G., Koeppel, R. 2014. Ring trial validation of single and multiplex real-time PCR methods for the detection and quantification of the allergenic food ingredients sesame, almond, lupine and Brazil nut. Journal of Consumer Protection and Food Safety, February 2014, [online] https://doi.org/10.1007/s00003-014-0868-x DOI: https://doi.org/10.1007/s00003-014-0868-x

Ziobro, R., Witczak, T., Juszczak, L., Korus, J. 2013. Supplementation of gluten-free bread with non-gluten proteins. Effect on dough rheological properties and bread characteristic. Food Hydrocolloids, vol. 32, no. 2, p. 213-220. https://doi.org/10.1016/j.foodhyd.2013.01.006 DOI: https://doi.org/10.1016/j.foodhyd.2013.01.006

Downloads

Published

2014-07-10

How to Cite

Revák, O. ., Golian, J. ., Židek, R. ., Čapla, J. ., & Zajác, P. . (2014). Detection of Lupine (Lupinus spp. L.) as a food allergen using three methods: end-point PCR, Real-Time PCR and Elisa. Potravinarstvo Slovak Journal of Food Sciences, 8(1), 207–215. https://doi.org/10.5219/384

Most read articles by the same author(s)

<< < 1 2 3 4 5 6 7 > >>