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The production and consumption of gluten-free food products have significantly increased due to the global rise in celiac disease and non-celiac gluten sensitivity. Ensuring the safety of these products remains a critical challenge, as accidental cross-contamination can occur during manufacturing, storage, or transport.
While conventional detection relies heavily on immunochemical methods like ELISA, these techniques face limitations when analyzing highly processed food matrices. Molecular DNA-based methods, particularly PCR combined with High-Resolution Melting (PCR-HRM), offer an alternative approach for the detection and discrimination of target cereal species. The aim of this study was to develop and evaluate specific PCR-HRM primers targeting the internal transcribed spacer (ITS) region for the discrimination of gluten-containing cereals from gluten-free cereals. Three primer sets (P1, P2, P3) were designed and experimentally evaluated for their specificity toward gluten-containing cereals, including wheat, barley, and rye, and their ability to discriminate them from gluten-free cereals, including oat, rice, and maize.
The first primer pair (P1) demonstrated insufficient specificity, as amplification was also observed in non-target cereal samples, limiting its ability to reliably discriminate gluten-containing from gluten-free cereals. The second primer pair (P2) improved performance but still exhibited non-specific amplification in oat samples. In contrast, the third primer set (P3) demonstrated high specificity, with clear discrimination between gluten-containing and gluten-free cereal samples. Primer performance was further evaluated by comparing amplification cycle threshold (Ct) values and the shape and characteristics of the amplification and melting curves. The selected primer set produced distinct PCR-HRM profiles: Tm in the range of 87.8 – 89.1 for wheat, barley and rye, Tm in the range of 77.9 – 81.6 for oat, rice and maize.
These results demonstrate that careful primer design and selection within the ITS region can provide a specific PCR-HRM system for the differentiation of gluten-containing and gluten-free cereals. Further optimization and validation using a broader range of reference materials and processed food matrices will be performed to determine the analytical sensitivity, detection limit, and applicability of the developed method.
Keywords: Gluten-containing cereals, PCR-HRM, ITS region, Primer specificity, Food safety