1. 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 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) StepTemperatureDurationRepeats Initial polymerase activation 95°C 15 min1 Denaturation 95°C 30 sec 35 Annealing 50°C 30 sec Elongation 72°C 30 sec Final elongation72°C5 min1 StepTemperatureDurationRepeats Initial polymerase activation 95°C 15 min1 Denaturation 95°C 30 sec 20 Annealing 65°C to 54°C 30 sec Elongation 72°C 30 sec Denaturation 95°C 30 sec 20 Annealing 54°C 30 sec Elongation 72°C 30 sec Final elongation72°C5 min1

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 Brazil nut 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