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 food fraud in cereal products, a significant component of human diet. In this work, primer–probe systems for real-time PCR and droplet digital PCR (ddPCR) for the detection of these cereal species: bread wheat (together with spelt), durum wheat, rye and barley for real-time PCR and ddPCR were established, optimized and validated. In addition, it was projected to validate a molecular system for differentiation of bread wheat and spelt; however, attempts for molecular differentiation between common wheat and spelt based on the gene GAG56D failed because of the genetic variability of the molecular target. Primer–probe systems were further developed and optimized on the basis of alignments of DNA sequences, as well as already developed PCR systems. The specificity of each system was demonstrated on 10 (spelt), 11 (durum wheat and rye) and 12 (bread wheat) reference samples. Specificity of the barley system was already proved in previous work. The calculated limits of detection (LOD95%) were between 2.43 and 4.07 single genome copies in real-time PCR. Based on the “three droplet rule”, the LOD95% in ddPCR was calculated to be 9.07–13.26 single genome copies. The systems were tested in mixtures of flours (rye and common wheat) and of semolina (durum and common wheat). The methods proved to be robust with regard to the tested conditions in the ddPCR. The developed primer–probe systems for ddPCR proved to be effective in quantitatively detecting the investigated cereal species rye and common wheat in mixtures by taking into account the haploid genome weight and the degree of milling of a flour. This method can correctly detect proportions of 50%, 60% and 90% wholemeal rye flour in a mixture of wholemeal common wheat flour. Quantitative results depend on the DNA content, on ploidy of cereal species and are also influenced by comminution. Hence, the proportion of less processed rye is overestimated in higher processed bread wheat and adulteration of durum wheat by common wheat by 1–5% resulted in underestimation of common wheat.

Vibrio-Sequins - dPCR-traceable DNA standards for quantitative genomics of Vibrio spp

“…1 ). In contrast to previous studies utilising more established dPCR technologies than the Stilla Naica crystal dPCR system (e.g., Biorad, Qiagen) [ 36 , 37 , 49 , 50 ], we report slightly elevated values for LODs between 10 and 26 cp/µl and consequently for LOQs between 23 and 124 cp/µl (Fig. 1 ).…”

Traceability of the local cultivar ‘Caaveiro’ in flour mixtures used to produce Galician bread by simple sequence repeats and droplet digital polymerase chain reaction technology

“…No previous references were found that discussed the use of SSRs in ddPCR for traceability and quantification of autochthonous cereal cultivars in flour blends, and a few were found based on SNPs and species‐specific sequences (Perry & Lee, 2017; Morcia et al ., 2020; Schulze et al ., 2021).…”

“…Quantitative estimates of wheat adulteration were found to depend on the DNA content and ploidy of cereal species and were also influenced by comminution, which is caused by overestimation of the proportion of less processed rye in more processed bread wheat and adulteration of durum wheat by common wheat by 1%-5%, resulting in underestimation of common wheat (Schulze et al, 2021), which we believe did not affect our results because, if this process was present, we knew the total amplicons for the pure flours for an accurate estimation.…”

“…Primer‐probe systems for real‐time PCR and droplet digital PCR (ddPCR) have been used previously for the detection of cereal species (Schulze et al ., 2021). ddPCR was effective in detecting and quantifying species of rye and common wheat in mixtures, but the results depended on the DNA content, and the ploidy of cereal species and were also influenced by comminution.…”