title: Detection of almond allergen coding sequences in processed foods by real time PCR.
abstract: The aim of this work was to develop and analytically validate a quantitative RT-PCR method, using novel primer sets designed on Pru du 1, Pru du 3, Pru du 4, and Pru du 6 allergen-coding sequences, and contrast the sensitivity and specificity of these probes. The temperature and/or pressure processing influence on the ability to detect these almond allergen targets was also analyzed. All primers allowed a specific and accurate amplification of these sequences. The specificity was assessed by amplifying DNA from almond, different Prunus species and other common plant food ingredients. The detection limit was 1 ppm in unprocessed almond kernels. The method's robustness and sensitivity were confirmed using spiked samples. Thermal treatment under pressure (autoclave) reduced yield and amplificability of almond DNA; however, high-hydrostatic pressure treatments did not produced such effects. Compared with ELISA assay outcomes, this RT-PCR showed higher sensitivity to detect almond traces in commercial foodstuffs.
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.forename: Elisa
authors.initials: E
authors.lastname: Iniesto
authors.forename: Carmen
authors.initials: C
authors.lastname: Burbano
authors.forename: Beatriz
authors.initials: B
authors.lastname: Cabanillas
authors.forename: Mercedes M
authors.initials: MM
authors.lastname: Pedrosa
authors.forename: Mercè
authors.initials: M
authors.lastname: Rovira
authors.forename: Julia
authors.initials: J
authors.lastname: Rodríguez
authors.forename: Mercedes
authors.initials: M
authors.lastname: Muzquiz
authors.forename: Jesus F
authors.initials: JF
authors.lastname: Crespo
authors.forename: Carmen
authors.initials: C
authors.lastname: Cuadrado
authors.forename: Rosario
authors.initials: R
authors.lastname: Linacero
mesh_terms: D000485:Allergens / Q000032:analysis*; D052179:Antigens, Plant / Q000276:immunology / Q000302:isolation & purification; D003001:Cloning, Molecular; D017931:DNA Primers; D018744:DNA, Plant / Q000235:genetics; D005511:Food Handling / Q000379:methods*; D027861:Prunus / Q000737:chemistry*; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA
chemical_list: D000485:Allergens; D052179:Antigens, Plant; D017931:DNA Primers; D018744:DNA, Plant