title: Detection by real time PCR of walnut allergen coding sequences in processed foods.
abstract: A quantitative real-time PCR (RT-PCR) method, employing novel primer sets designed on Jug r 1, Jug r 3, and Jug r 4 allergen-coding sequences, was set up and validated. Its specificity, sensitivity, and applicability were evaluated. The DNA extraction method based on CTAB-phenol-chloroform was best for walnut. RT-PCR allowed a specific and accurate amplification of allergen sequence, and the limit of detection was 2.5pg of walnut DNA. The method sensitivity and robustness were confirmed with spiked samples, and Jug r 3 primers detected up to 100mg/kg of raw walnut (LOD 0.01%, LOQ 0.05%). Thermal treatment combined with pressure (autoclaving) reduced yield and amplification (integrity and quality) of walnut DNA. High hydrostatic pressure (HHP) did not produce any effect on the walnut DNA amplification. This RT-PCR method showed greater sensitivity and reliability in the detection of walnut traces in commercial foodstuffs compared with ELISA assays.
authors.affiliation: Departamento de Genética, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Rosario
authors.initials: R
authors.lastname: Linacero
authors.affiliation: Departamento de Tecnología de Alimentos, SGIT-INIA, Ctra. La Coruña km. 7.5, 28040 Madrid, Spain.
authors.forename: Isabel
authors.initials: I
authors.lastname: Ballesteros
authors.affiliation: Departamento de Tecnología de Alimentos, SGIT-INIA, Ctra. La Coruña km. 7.5, 28040 Madrid, Spain.
authors.forename: Africa
authors.initials: A
authors.lastname: Sanchiz
authors.affiliation: Departamento de Genética, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Nuria
authors.initials: N
authors.lastname: Prieto
authors.affiliation: Departamento de Genética, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Elisa
authors.initials: E
authors.lastname: Iniesto
authors.affiliation: Departamento de Genética, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain.
authors.forename: Yolanda
authors.initials: Y
authors.lastname: Martinez
authors.affiliation: Departamento de Tecnología de Alimentos, SGIT-INIA, Ctra. La Coruña km. 7.5, 28040 Madrid, Spain.
authors.forename: Mercedes M
authors.initials: MM
authors.lastname: Pedrosa
authors.affiliation: Departamento de Tecnología de Alimentos, SGIT-INIA, Ctra. La Coruña km. 7.5, 28040 Madrid, Spain.
authors.forename: Mercedes
authors.initials: M
authors.lastname: Muzquiz
authors.affiliation: Servicio de Alergia, Instituto de Investigación Hospital 12 de Octubre (i+12), Avenida de Córdoba s/n, 28041 Madrid, Spain.
authors.forename: Beatriz
authors.initials: B
authors.lastname: Cabanillas
authors.affiliation: Institut de Recerca i Tecnologia Agroalimentàries (IRTA)-Mas de Bover, Ctra. Reus-El Morell km 3.8, 43120, Spain.
authors.forename: Mercè
authors.initials: M
authors.lastname: Rovira
authors.affiliation: Departamento de Tecnología de Alimentos, SGIT-INIA, Ctra. La Coruña km. 7.5, 28040 Madrid, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Burbano
authors.affiliation: Departamento de Tecnología de Alimentos, SGIT-INIA, Ctra. La Coruña km. 7.5, 28040 Madrid, Spain. Electronic address: cuadrado@inia.es.
authors.forename: Carmen
authors.initials: C
authors.lastname: Cuadrado
mesh_terms: D000485:Allergens / Q000032:analysis* / Q000276:immunology; D052179:Antigens, Plant / Q000032:analysis* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005504:Food Analysis / Q000379:methods*; D031324:Juglans / Q000235:genetics* / Q000276:immunology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D000485:Allergens; D052179:Antigens, Plant
keywords: Juglans regia; Pressure processing; Processed foods; Real-time PCR; Thermal processing; Walnut allergen detection