Development and validation of a method for quantification of common wheat, durum wheat, rye and barley by droplet digital PCR

Abstract Food fraud is becoming a prominent topic in the food industry. Thus, valid methods for detecting potential adulterations are necessary to identify instances of cereal products, significant component human diet. In this work, primer–probe systems real-time PCR and droplet digital (ddPCR) detection these species: bread wheat (together with spelt), durum wheat, rye barley ddPCR were established, optimized validated. addition, it was projected validate molecular system differentiation spelt; however, attempts between common spelt based on gene GAG56D failed because genetic variability target. Primer–probe further developed basis alignments DNA sequences, as well already systems. The specificity each demonstrated 10 (spelt), 11 (durum rye) 12 (bread wheat) reference samples. Specificity proved previous work. calculated limits (LOD 95% ) 2.43 4.07 single genome copies PCR. Based “three rule”, LOD be 9.07–13.26 copies. tested mixtures flours (rye semolina wheat). robust regard conditions ddPCR. effective quantitatively investigated species by taking into account haploid weight degree milling flour. This method can correctly detect proportions 50%, 60% 90% wholemeal flour mixture Quantitative results depend content, ploidy also influenced comminution. Hence, proportion less processed overestimated higher adulteration 1–5% resulted underestimation wheat.