A real-time PCR method for the detection of pine nuts in food.

Pine nuts, worldwide consumed as such, or as ingredient in desserts, pastries, sauces (Italian pesto) or salads, is also known to be a source of food allergens. Several allergic reactions (in most cases anaphylaxis) referred to pine nuts have been reported, after ingestion or by simple inhalation [1,2]. In order to protect allergic consumers, methods to unequivocally detect the presence of pine nuts in complex food matrices need to be developed. A Taqman-based real-time PCR method for the detection of Pinus spp. DNA was set up. Genomic DNA was isolated from pine nuts purchased in local markets, from pine needles belonging to nine different species of pine tree producing edible nuts, and from other sources commonly employed in the preparation of pine nut-containing foods. Moreover, model foods spiked at known concentration of pine nut powder were prepared in lab. Finally, DNA was purified from commercial foods declaring (or not) the presence of pine nuts. Selected primer pairs and probe displayed an efficiency near to 100% when applied to DNA isolated from pine nut seeds. Eight out of nine species of Pinus employed in the specificity assay were positively amplified, while none of the 18 negative controls displayed amplification. Both the intrinsic detection limit and the practical one evaluated on model spiked foods was similar or higher than that of other commercially available detection kit. Finally, the presence/absence of pine nut DNA in commercial foods was confirmed by our method.