DNA-BASED DETECTION METHOD OF PLANT-DERIVED ALLERGENS IN FOODSTUFFS Master's Thesis Jekaterina Losseva Supervisors: Olga Bragina, PhD; TUT Svetlana Sergejeva,

DNA-BASED DETECTION DNA-BASED DETECTION METHODMETHODOF PLANT-DERIVED ALLERGENS OF PLANT-DERIVED ALLERGENS IN FOODSTUFFSIN FOODSTUFFSMaster's Thesis

Jekaterina LossevaSupervisors: Olga Bragina, PhD; TUT

Svetlana Sergejeva, MD, PhD; TUIT

Lilian Järvekülg, PhD; TUT

11.06.2014

The planThe planIntroduction

◦Food hypersensitivity◦Food allergy◦Detection methods

Aims of the studyMaterials and methodsResults

Food hypersensitivityFood hypersensitivity

IgE-mediated allergic IgE-mediated allergic reactionreaction

Food allergyFood allergyPrevalence

◦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 allergensDetection of allergensProtein-based methods

◦Immunoblotting◦enzyme-linked immunosorbent assay

(ELISA)DNA-based methods

◦PCR◦PCR-ELISA◦Real-time PCR

Aims of this studyAims of this studydetermine 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 methodsMaterials and methodsDNA extraction methods

◦Basic guanidine/chloroform◦Basic phenol/chloroform◦Cetyltrimethylammonium bromide

(CTAB)◦CTAB Precipitation◦2xCTAB◦DNeasy Plant Mini Kit◦Some modifications to the Kit

Materials and methodsMaterials and methodsPCR amplification

Universal primers

Species-specific primers (Touchdown PCR)

Step Temperature Duration Repeats

Initial polymerase activation 95°C 15 min 1

Denaturation 95°C 30 sec

35Annealing 50°C 30 sec

Elongation 72°C 30 sec

Final elongation 72°C 5 min 1

Step Temperature Duration Repeats

Initial polymerase activation 95°C 15 min 1

Denaturation 95°C 30 sec

20Annealing 65°C to 54°C 30 sec

Elongation 72°C 30 sec

Denaturation 95°C 30 sec

20Annealing 54°C 30 sec

Elongation 72°C 30 sec

Final elongation 72°C 5 min 1

DNA extraction methodsDNA 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 methodsDNA extraction methods 2xCTAB

DNA extraction methodsDNA extraction methods DNeasy Plant Mini Kit

DNA extraction methodsDNA extraction methods DNeasy Plant Mini Kit with modifications

Universal primersUniversal primers P-Uni1 B – P-Uni2 C – P-Uni3 P-Uni4 P-Uni5 F – P-Uni6

Species-specific primersSpecies-specific primersMacadamiaHazelnut 0,025 ngPeanutPistachio 0,025 ngAlmond 0,0025 ngWalnutCashew 0,25 ngBrazil nut 0,0025

ngPecan

Soya 0,025 ngMustard orientalMustard whiteSesame 0,025 ngLupine 0,0025 ngWheat 0,0025 ngRyeBarley 0,0025 ngOat 0,0025 ngCelery

Species-specific primersSpecies-specific primers A – Hazelnut B – Pistachio

Species-specific primersSpecies-specific primers B, C – Sesame Ses1,

Ses2

Non-specific primersNon-specific primers Peanut

SummarySummaryDNA 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

AcknowledgementsAcknowledgementsOlga BraginaJelena TsõmbalovaSvetlana SergejevaBerit PildenLilian Järvekülg