High resolution melting analysis as a new approach to discriminate gluten-containing cereals.
With this work, it is intended to propose a novel approach based on high resolution melting (HRM) analysis to detect wheat and discriminate it from other gluten-containing cereals. The method consisted of a real-time PCR assay targeting the gene encoding for the germ agglutinin isolectin A protein (Tri a 18 allergen), using the fluorescent Evagreen dye combined with HRM analysis. The results enabled wheat differentiation from other phylogenetically related cereals, namely barley, rye and oat with high level of confidence. Additionally, a quantitative real-time PCR approach was proposed, allowing detecting and quantifying wheat down to 20mg/kg in rice flour and 20pg of wheat DNA (∼1.1 DNA copies). Its application was successfully achieved in the analysis of processed foods to verify labelling compliance, being considered as a cost-effective tool for the specific detection of cereals in gluten-free foods.
REQUIMTE-LAQV, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal; Sección Departamental de Química Analítica, Facultad de Farmacia, Universidad Complutense de Madrid, Pz. Ramóny Cajal s/n, 28040 Madrid, Spain.
Begoña
B
Martín-Fernández
REQUIMTE-LAQV, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
Joana
J
Costa
Departamento Química Física y Analítica, Universidad de Oviedo, Av. Julián Claveria 8, 33006 Oviedo, Spain.
Noemí
N
de-Los-Santos-Álvarez
Sección Departamental de Química Analítica, Facultad de Farmacia, Universidad Complutense de Madrid, Pz. Ramóny Cajal s/n, 28040 Madrid, Spain.
Beatriz
B
López-Ruiz
REQUIMTE-LAQV, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal.
M Beatriz P P
MB
Oliveira
REQUIMTE-LAQV, Faculdade de Farmácia, Universidade do Porto, Rua de Jorge Viterbo Ferreira, 228, 4050-313 Porto, Portugal. Electronic address: isabel.mafra@ff.up.pt.
Isabel
I
Mafra
D000485:Allergens / Q000032:analysis* / Q000235:genetics; D001483:Base Sequence; D002523:Edible Grain / Q000737:chemistry* / Q000235:genetics; D005433:Flour / Q000032:analysis; D005983:Glutens / Q000032:analysis* / Q000235:genetics; D001467:Hordeum / Q000737:chemistry / Q000235:genetics; D012275:Oryza / Q000737:chemistry / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D014908:Triticum / Q000737:chemistry / Q000235:genetics
D000485:Allergens; D005983:Glutens
Agglutinin isolectin; Celiac disease; Gluten-containing cereals; Real-time PCR; Wheat; Wheat allergy