Real-time PCR systems for the detection of the gluten-containing cereals wheat, spelt, kamut, rye, barley and oat

Real-time PCR assays, using TaqMan® probes, were applied to detect the gluten-containing cereals. Homologues target sequences encoding high molecular weight (HMW) glutenin were chosen to detect wheat, kamut, spelt and rye. For detecting barley, the gene Hor3 was selected. For the detection of oat the gene encoding the 12S seed storage protein was chosen. Based on this sequence data, primer and probe sequences were generated for the real-time PCR. A plant specific primer probe system based on 18S rRNA gene was chosen to detect amplificability of the extracted nucleic acids. The specificity of the primer and probe systems was checked using different lines from different origins of the species to be detected. The HMW glutenin system is specific for the corresponding species, as are the systems for the barley Hor3 gene and the oat 12S seed storage protein. The sensitivity of the systems was determined testing different matrices. With the HMW glutenin system 2.5 mg/kg of wheat in vegetable food matrices and 5 mg/kg of wheat in meat products were detected. The oat and the barley specific systems resulted in a sensitivity of 10 mg/kg. The detection method showed a satisfactory ruggedness.