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