title: Tracing tree nut allergens in chocolate: A comparison of DNA extraction protocols.
abstract: The present work aimed at comparing different DNA extraction methods, from chocolate matrices, for the effective application in molecular techniques to detect tree nut allergens. For this study, DNA from almond or hazelnut model chocolates was extracted using seven selected protocols: the in-house methods of CTAB-PVP (cetyltrimethylammonium bromide-polyvinylpyrrolidone), Wizard with and without RNase, Wizard-PVP with and without RNase, and the Wizard Magnetic and Nucleospin kits. The extracts were assessed for their suitability for amplification by qualitative PCR and real-time PCR. From the evaluated protocols, Nucleospin presented the best results for almond and hazelnut amplification, achieving a limit of detection of 0.005% (w/w) with high PCR efficiency, linearity and range of amplification. These results highlight the importance of the DNA extraction protocol in the case of food allergens from complex matrices, such as chocolate, in which sensitivity is a key parameter.
authors.affiliation: REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Electronic address: joanabcosta@gmail.com.
authors.forename: Joana
authors.initials: J
authors.lastname: Costa
authors.affiliation: REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: Vítor S
authors.initials: VS
authors.lastname: Melo
authors.affiliation: REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: Cristina G
authors.initials: CG
authors.lastname: Santos
authors.affiliation: REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
authors.forename: M Beatriz P P
authors.initials: MB
authors.lastname: Oliveira
authors.affiliation: REQUIMTE, Departamento de Ciências Químicas, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Electronic address: isabel.mafra@ff.up.pt.
authors.forename: Isabel
authors.initials: I
authors.lastname: Mafra
mesh_terms: D000485:Allergens / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D002099:Cacao / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D031211:Corylus / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification*; D009754:Nuts / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D060888:Real-Time Polymerase Chain Reaction
chemical_list: D000485:Allergens; D018744:DNA, Plant
keywords: Almond; Chocolate; DNA extraction; Food allergens; Hazelnut; Real-time PCR