Digital PCR For the Quantification of Common Wheat Content in Spelt Products

Digital PCR For the Quantification of Common Wheat Content in Spelt Products

Abstract
Common wheat (Triticum aestivum ssp. aestivum) and spelt (Triticum aestivum ssp. spelta) are two closely related hexaploid cereals of the Triticum genus. Spelt is considered an original, natural and undemanding crop and consumers often prefer spelt over common wheat due to its higher nutritional value and taste. Some consumers also reported a better tolerance in terms of digestion and food allergy. World market prices for spelt have soared based on the high demand, making spelt products a worthwhile target for food fraud.
Due to their close genetic relationship, it is analytically difficult to specifically distinguish common wheat and spelt on DNA level. Spelt product adulteration with common wheat can be detected using qPCR, however, crossbreading lead to some spelt cultivars with a recombined genetic structure. Those cultivars would be analytically determined as common wheat with the present qPCR methods. Furthermore, none of the published qPCR methods focuses on a quantitative analysis.
Hence, we developed a duplex digital droplet PCR (ddPCR) method for the quantitative determination of the common wheat content in spelt products by targeting two genetic differences: a single nucleotide variant in the Q-locus (FAM channel) and a 9 bp deletion in the γ-gliadin gene (HEX channel; same target as in qPCR). The hydrolysis probes for both assays were designed to match the spelt specific DNA sequence. In the hydrolysis probe of the Q-locus assay, we added an additional mismatch for spelt and common wheat to destabilize the probe. As a consequence, binding of the probe in both assays (Q-locus, γ-gliadin) is better for spelt DNA compared to common wheat DNA. In ddPCR, this results in three distinct droplet populations, a negative population (lowest fluorescence signal), a common wheat positive population (lower fluorescence signal) and a spelt positive population (highest fluorescence signal). By different positioning of the threshold, the DNA copy number concentrations of spelt and spelt+wheat can now be determined separately. The spelt to common wheat ratio is then calculated based on these concentrations (ratio = 100 % × DNA copy number concentration spelt / DNA copy number concentration spelt+wheat).
In-house validation and interlaboratory tests with different matrices were performed to verify the sensitivity, precision and robustness of the method. Specificity of the methods was demonstrated by analyzing 84 spelt cultivars, 73 common wheat cultivars, 19 einkorn wheat cultivars (Triticum monococcum), 18 emmer cultivars (Triticum dicoccon), 4 durum wheat cultivars (Triticum durum), 8 oat cultivars (Avena spp.) and 7 rye cultivars (Secale cereale). In the second half of 2022, a ring-trial was conducted by the German § 64 LFGB working group “species identification” to validate this method.