Development of molecular markers for detecting almond, peanut, pine nut, and walnut in commercial food using quantitative real-time PCR

Nuts have been used globally as health foods. However, because nuts cause allergies, people need to be careful when eating food. Mostly foods are labeled, but sometimes intentional or unintentional mixing might occur. In the present study, we report DNA based on marker for the detection of four nuts almond, peanut, pine nut, and walnut using quantitative real-time polymerase chain reaction (qRT-PCR). Species-specific primer sets for four species were designed based on the single-nucleotide polymorphisms and insertion/deletion of the chloroplast gene, matK. The sensitivity of primer sets for the four species studied was assessed by analyzing DNA dilutions at concentration of 0.001–10 ng and binary mixtures of 0.1–100% of heat-treated and non-heat-treated samples. The four primer sets developed in the present study indicated appropriate amplification efficiency and correlation coefficients of the standard curves. In addition, to verify the applicability of these molecular markers, we performed a qRT-PCR with 14 commercial products and successfully detected the matK genes in several commercial food products that were declared to contain nuts. Thus, markers developed could be useful tools for confirming the presence of the four nut species in commercial products.