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
The plan

Introduction
◦ Food hypersensitivity
◦ Food allergy
◦ Detection methods
Aims of the study
 Materials and methods
 Results

Food hypersensitivity
IgE-mediated allergic reaction
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.
Detection of allergens

Protein-based methods
◦ Immunoblotting
◦ enzyme-linked immunosorbent assay (ELISA)

DNA-based methods
◦ PCR
◦ PCR-ELISA
◦ Real-time PCR
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.

Materials and methods

DNA extraction methods
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◦
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Basic guanidine/chloroform
Basic phenol/chloroform
Cetyltrimethylammonium bromide (CTAB)
CTAB Precipitation
2xCTAB
DNeasy Plant Mini Kit
Some modifications to the Kit
Materials and methods

PCR amplification
 Universal primers
Step
Temperature
Duration
Repeats
Initial polymerase activation
95°C
15 min
1
Denaturation
95°C
30 sec
Annealing
50°C
30 sec
Elongation
72°C
30 sec
Final elongation
72°C
5 min
35
1
 Species-specific primers (Touchdown PCR)
Step
Temperature
Duration
Repeats
Initial polymerase activation
95°C
15 min
1
Denaturation
95°C
30 sec
Annealing
65°C to 54°C
30 sec
Elongation
72°C
30 sec
Denaturation
95°C
30 sec
Annealing
54°C
30 sec
Elongation
72°C
30 sec
Final elongation
72°C
5 min
20
20
1
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
DNA extraction methods
 2xCTAB
DNA extraction methods
 DNeasy Plant Mini Kit
DNA extraction methods
 DNeasy Plant Mini Kit with modifications
Universal primers
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P-Uni1
B – P-Uni2
C – P-Uni3
P-Uni4
P-Uni5
F – P-Uni6
Species-specific primers

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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
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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
Species-specific primers
 A – Hazelnut
 B – Pistachio
Species-specific primers
 B, C – Sesame Ses1, Ses2
Non-specific primers
 Peanut
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
Acknowledgements
Olga Bragina
 Jelena Tsõmbalova
 Svetlana Sergejeva
 Berit Pilden
 Lilian Järvekülg
