Nowadays the design of modern technologies of production of specialized nutrition products is actual enough. Non-gluten products for people who has gluten entheropathy belong to this type of products. Celiac disease (gluten enteropathy) is an autoimmune hereditary disease and is characterized by persistent intolerance of gluten in such cereal as wheat, rye, barley and less oat. This is widespread disease, quantity of celiac-disease people is in range of 0,5–1%. Non-gluten diet during the whole life is the only method of cure and prevention of celiac-aftereffects. Modern high-sensitive and low-expensive test-systems are necessary for appropriate control of non-gluten foodstuffs.

The aim of current study was the design of cereal gluten detection method and development and approbation of domestic real-time PCR-based test-system for gluten detection and identification in foodstuffs.

Samples of wheat, rye, barley, oat and other cereals seeds, foodstuffs and raw material were matrices for genomic DNA isolation. DNA was extracted by CTAB-precipitation method with own modification. Glu B1-1 of wheat and Glu1-R of rye were target genes for gluten detection. Hor3 and 12S SSP genes were targets for barley and oat detection. TaqMan technology with it high sensitivity, specificity and rapidity of performance was used for test-system design. PCR conditions were as follows: 100 ng DNA, 10 mM Tris-HCl (pH 8,3), 50 mM KCl, 2,5 mM MgCl₂, 0,2 mM dNTP-mix, 20 pkM each primers, 10 pkM probe and 1 U of Taq DNA polymerase (Thermo Scientific™, Lithunia). The applied cycles were: denaturation
at 95 ºС/3 min, followed by 45 cycles of 95 ºС/15 s, annealing and synthesis at 60 or 65 ºС /40 s.

Proposed test-system is multiplex and consists of two PCR-mixes. One PCR-mix allows wheat, rye and oat DNA identification, another – for barley and psbA gene DNA identification. The latter was chosen as the species-specific PCR endogenous control. Behavior of each reaction was monitored by using of fluorescent-labeled probes FAM (12S SSP), ROX (Hor3), HEX (Glu1-R, psbA) and Cy5 (Glu B1-1).

Optimization of amplification was performed for primers annealing temperature, MgCl₂ concentration, concentration and ratio of primers and probes. The optimum values of concentrations was 2,5 мМ for MgCl₂, 20 pkM for primers and 10 pkM for probes. Effectiveness of test-system was performed for specificity, sensitivity, repeatability and reproducibility of results according to PCR test-systems design.

Secale cereal, Triticum aestivum, Hordeum vulgare, Avena sativa, Fagopyrum esculentum, Oryza sativa, Zea mays, Glycine max, Brassica napus, Lycopersican esculentum, Solanum tuberosum were tested for specificity detection. Crossed reactions were not revealed.

The sensitivity limit of developed test-system was detected by preparation of series of 10x DNA dilutions from 0,0001 до 100 ng of genomic DNA. Sensitivity limit of system for genes Glu B1-1, Glu1-R, Hor3 and 12S SSP was 0,1 ng, for gene psbA – 0,001 ng. Our results agreed with data obtained by other authors and by use of commercial test-systems.

Test-system approbation was conducted during 2013–2015 by comparative tests of foodstuffs with known content. Commercial ELISA and PCR test-systems RIDASCREEN® Gliadin and SureFood® ALLERGEN ID Gluten (R-Biopharm, Germany) were used for comparison. 53 foodstuffs and raw material samples were tested totally. Obtained data confirmed comparability of the developed test-system. Among 53 tested samples 43 (81 %) were non-gluten (< 5 mg/kg). In 10 (19%) remained samples gluten was identified from 4,6 mg/kg and more. Most of samples tested were not labeled as non-gluten products so the data has only informative effect.

Conducted analysis confirmed the availability of developed test-system for testing and detection of wheat, rye, barley and oat in raw material (grain, flour), and in processed products (cereals, cookies, malt extracts). Besides, sensitivity of test-system allows the identification of so called «hidden» gluten, which can be found in products of industrial production as admixes-stiffeners, form-building agents, stabilizers, wheat flour, starch etc.  

Key words: gluten, celiac disease, Real-Time PCR, food products.