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DNA-based detection method of plant-derived allergens in foodstuffs

DNA-based detection method of plant-derived allergens in foodstuffs. Master's Thesis Jekaterina Losseva Supervisors: Olga Bragina , PhD; TUT Svetlana Sergejeva , MD, PhD; TUIT Lilian Järvekülg , PhD; TUT. The plan. Introduction Food hypersensitivity Food allergy Detection methods

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DNA-based detection method of plant-derived allergens in foodstuffs

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  1. DNA-based detection methodof plant-derived allergens in foodstuffs Master's Thesis JekaterinaLosseva Supervisors: Olga Bragina, PhD; TUT Svetlana Sergejeva, MD, PhD; TUIT LilianJärvekülg, PhD; TUT 11.06.2014

  2. The plan • Introduction • Food hypersensitivity • Food allergy • Detection methods • Aims of the study • Materials and methods • Results

  3. Food hypersensitivity

  4. IgE-mediated allergic reaction

  5. Food allergy • Prevalence • 2% - adults • 18% - children • Treatment • Avoidance of the causative food • Potentially allergenic foods • gluten-containing cereals, crustaceans, eggs, fish, nuts, soybean, milk, celery, mustard, sesame, sulphites, lupine and molluscs.

  6. Detection of allergens • Protein-based methods • Immunoblotting • enzyme-linked immunosorbent assay (ELISA) • DNA-based methods • PCR • PCR-ELISA • Real-time PCR

  7. Aims of this study • determine the most sufficient plant DNA extraction methods from different foodstuffs • create DNA library of selected plants • evaluate specie-specificity of designed in the frame of Allergofood project primers • examine the sensitivity of species-specific primers.

  8. Materials and methods • DNA extraction methods • Basic guanidine/chloroform • Basic phenol/chloroform • Cetyltrimethylammonium bromide (CTAB) • CTAB Precipitation • 2xCTAB • DNeasy Plant Mini Kit • Some modifications to the Kit

  9. Materials and methods • PCR amplification • Universal primers • Species-specific primers (Touchdown PCR)

  10. DNA extraction methods • A – basic phenol/chloroform • C – CTAB • B – basic guanidine/chloroform • D – CTAB precipitation Lanes 2 to 9 represent mustard, cacao, Brazil nut, pecan, chocolate with plum, chocolate with cherry, sesame and radish

  11. DNA extraction methods • 2xCTAB

  12. DNA extraction methods • DNeasy Plant Mini Kit

  13. DNA extraction methods • DNeasy Plant Mini Kit with modifications

  14. Universal primers • P-Uni1 • B – P-Uni2 • C – P-Uni3 • P-Uni4 • P-Uni5 • F – P-Uni6

  15. Species-specific primers • Macadamia • Hazelnut 0,025 ng • Peanut • Pistachio 0,025 ng • Almond 0,0025 ng • Walnut • Cashew 0,25 ng • Brazilnut 0,0025 ng • Pecan • Soya 0,025 ng • Mustard oriental • Mustard white • Sesame 0,025 ng • Lupine 0,0025 ng • Wheat 0,0025 ng • Rye • Barley 0,0025 ng • Oat 0,0025 ng • Celery

  16. Species-specific primers • A – Hazelnut • B – Pistachio

  17. Species-specific primers • B, C – Sesame Ses1, Ses2

  18. Non-specific primers • Peanut

  19. Summary • DNA extraction: • CTAB precipitation method for DNA extraction from foodstuffs • DNeasy Plant Mini Kit for DNA extraction from leaf • The same Kit with modification for DNA extraction from nuts • Touchdown PCR method as highly sensitive and specific method

  20. Acknowledgements • Olga Bragina • Jelena Tsõmbalova • Svetlana Sergejeva • Berit Pilden • Lilian Järvekülg

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