Use of multiplex ligation-dependent probe amplification (MLPA) for screening of wheat, barley, rye and oats in foods

A multiplex ligation-dependent probe amplification (MLPA) approach is described for the simultaneous detection of DNA from four common cereal ingredients in foods: wheat, barley, rye and oats. The method uses species-specific MLPA half-probes targeting DNA fragments from the ribosomal internal space transcriber (ITS) region for the detection of barley and oats, and from the genes encoding the low molecular weight (LMW) glutenin and the ω-secalin for wheat and rye, respectively. After hybridization, the probes are ligated and amplified by polymerase chain reaction (PCR) into specific and size-differential amplicons that are simultaneously detected by capillary electrophoresis. The cereal MLPA technique showed an optimal specificity against a representative number of plant and animal species. A sensitive detection of the targets (LOD of 50 mg kg−1) was achieved in a reference model cake experimentally spiked with different levels of each wheat, barley, rye and oats seeds. MLPA applicability was assessed through the analysis of 40 commercial food products with different labeling declarations regarding the targets (contain, may contain and do not declare/gluten free), indicating the presence of non-declared cereal ingredients in some of the tested samples. MLPA results were further confirmed by simplex Taqman real-time PCR assays. The described MLPA technique is a reliable and sensitive tool for screening the presence of low amounts of wheat, barley, rye and oats in processed foodstuffs, contributing to compliance with authenticity legal requirements and protecting consumers from fraudulent commercial practices or adventitious contamination which may lead to allergic reactions associated to cereal proteins ingestion.