Detection of gluten-containing cereals in food by 5-nuclease real-time polymerase chain reaction

A real-time polymerase chain reaction (PCR)-based method for the detection of coeliac disease-causing gluten-containing cereals (wheat, barley and rye) in food is described. The method consists of DNA isolation by chaotropic solid phase extraction and the subsequent real-time PCR with primers and a 5-nuclease (TaqMan) fluorescent probe targeted to the gene encoding for puroindoline b. The method produced positive results for 31 wheat cultivars as well as barley and rye samples, and negative ones for 18 other samples. The intrinsic detection limit of the method was 0.026 ng wheat DNA, which corresponds to approx. 1.5 haploid genomes. In model flour samples, 200 mg per kg of the wheat component could be detected, which was comparable to the detection limit of gliadin-targeting enzyme‑linked immunosorbent assay (ELISA). A linear calibration line was obtained for real-time PCR in the range from 200 mg per kg to 2000 mg per kg. Practical applicability of the real-time PCR method was tested by the analysis of 49 food samples, out of which 3 were found positive by both real‑time PCR and ELISA, and one sample was found positive by real-time PCR only. The presented real-time PCR is useful for sensitive and selective detection of coeliac disease-causing gluten-containing cereals in food products.