Tracing tree nut allergens in chocolate: A comparison of DNA extraction protocols. (15th November 2015)

• Record Type: Journal Article
• Authors: Costa, Joana; Melo, Vítor S.; Santos, Cristina G.; Oliveira, M. Beatriz P.P.; Mafra, Isabel
• Is Part Of: Food chemistry. Volume 187(2015)
• Page Start: 469
• Page End: 476
• Publication Date: 2015-11-15
• Subjects: Food allergens -- Almond -- Hazelnut -- Chocolate -- DNA extraction -- Real-time PCR; Food -- Analysis -- Periodicals; Food -- Composition -- Periodicals; 664
• DOI: 10.1016/j.foodchem.2015.04.073
• Languages: English
• ISSNs: 0308-8146

Abstract

Highlights: DNA extraction from almond or hazelnut in chocolate matrices. Comparison of several DNA extraction protocols. Evaluation of extracts regarding suitability for PCR amplification. Evaluation of performance for real-time PCR detection of allergens. Nucleospin presented the best results for reproducibility and sensitivity. 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.