title: Electrochemical detection of magnetically-entrapped DNA sequences from complex samples by multiplexed enzymatic labelling: Application to a transgenic food/feed quantitative survey.
abstract: Monitoring of genetically modified organisms in food and feed demands molecular techniques that deliver accurate quantitative results. Electrochemical DNA detection has been widely described in this field, yet most reports convey qualitative data and application in processed food and feed samples is limited. Herein, the applicability of an electrochemical multiplex assay for DNA quantification in complex samples is assessed. The method consists of the simultaneous magnetic entrapment via sandwich hybridisation of two DNA sequences (event-specific and taxon-specific) onto the surface of magnetic microparticles, followed by bienzymatic labelling. As proof-of-concept, we report its application in a transgenic food/feed survey where relative quantification (two-target approach) of Roundup Ready Soybean® (RRS) was performed in food and feed. Quantitative coupling to end-point PCR was performed and calibration was achieved from 22 and 243 DNA copies spanning two orders of magnitude for the event and taxon-specific sequences, respectively. We collected a total of 33 soybean-containing samples acquired in local supermarkets, four out of which were found to contain undeclared presence of genetically modified soybean. A real-time PCR method was used to verify these findings. High correlation was found between results, indicating the suitability of the proposed multiplex method for food and feed monitoring.
authors.affiliation: Sección Departamental de Química Analítica, Facultad de Farmacia, Universidad Complutense de Madrid, Pz. Ramón y Cajal s/n, 28040 Madrid, Spain; Departamento de Biotecnología - Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain.
authors.forename: C L
authors.initials: CL
authors.lastname: Manzanares-Palenzuela
authors.affiliation: Departamento de Biotecnología - Biología Vegetal, Escuela Técnica Superior de Ingeniería Agronómica, Alimentaria y de Biosistemas, Universidad Politécnica de Madrid, Ciudad Universitaria s/n, 28040 Madrid, Spain. Electronic address: juanpedro.martin@upm.es.
authors.forename: J P
authors.initials: JP
authors.lastname: Martín-Clemente
authors.affiliation: Departamento de Química Física y Analítica, Universidad de Oviedo, Av. Julián Clavería 8, 33006 Oviedo, Spain. Electronic address: mjlc@uniovi.es.
authors.forename: M J
authors.initials: MJ
authors.lastname: Lobo-Castañón
authors.affiliation: Sección Departamental de Química Analítica, Facultad de Farmacia, Universidad Complutense de Madrid, Pz. Ramón y Cajal s/n, 28040 Madrid, Spain. Electronic address: bealopru@ucm.es.
authors.forename: B
authors.initials: B
authors.lastname: López-Ruiz
mesh_terms: D000818:Animals; D001483:Base Sequence; D018744:DNA, Plant / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D004563:Electrochemistry / Q000379:methods*; D004798:Enzymes / Q000378:metabolism*; D059346:Magnets / Q000737:chemistry*; D008863:Microspheres; D030821:Plants, Genetically Modified; D016133:Polymerase Chain Reaction; D011795:Surveys and Questionnaires
chemical_list: D018744:DNA, Plant; D004798:Enzymes
keywords: DNA quantification; Electrochemical multiplex DNA detection; GMO quantification; Magnetic microparticles; Sample analysis