title: Targeting helicase-dependent amplification products with an electrochemical genosensor for reliable and sensitive screening of genetically modified organisms.
abstract: Cultivation of genetically modified organisms (GMOs) and their use in food and feed is constantly expanding; thus, the question of informing consumers about their presence in food has proven of significant interest. The development of sensitive, rapid, robust, and reliable methods for the detection of GMOs is crucial for proper food labeling. In response, we have experimentally characterized the helicase-dependent isothermal amplification (HDA) and sequence-specific detection of a transgene from the Cauliflower Mosaic Virus 35S Promoter (CaMV35S), inserted into most transgenic plants. HDA is one of the simplest approaches for DNA amplification, emulating the bacterial replication machinery, and resembling PCR but under isothermal conditions. However, it usually suffers from a lack of selectivity, which is due to the accumulation of spurious amplification products. To improve the selectivity of HDA, which makes the detection of amplification products more reliable, we have developed an electrochemical platform targeting the central sequence of HDA copies of the transgene. A binary monolayer architecture is built onto a thin gold film where, upon the formation of perfect nucleic acid duplexes with the amplification products, these are enzyme-labeled and electrochemically transduced. The resulting combined system increases genosensor detectability up to 10(6)-fold, allowing Yes/No detection of GMOs with a limit of detection of ∼30 copies of the CaMV35S genomic DNA. A set of general utility rules in the design of genosensors for detection of HDA amplicons, which may assist in the development of point-of-care tests, is also included. The method provides a versatile tool for detecting nucleic acids with extremely low abundance not only for food safety control but also in the diagnostics and environmental control areas.
authors.affiliation: †Departamento de Química Física y Analítica, Universidad de Oviedo, 33006 Oviedo, Spain.|‡Departamento de Química, Centro de Ciências da Natureza. Universidade Federal do Piauí, Teresina, 64049-550 PI, Brasil.
authors.forename: Suely
authors.initials: S
authors.lastname: Moura-Melo
authors.affiliation: †Departamento de Química Física y Analítica, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Rebeca
authors.initials: R
authors.lastname: Miranda-Castro
authors.affiliation: †Departamento de Química Física y Analítica, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Noemí
authors.initials: N
authors.lastname: de-Los-Santos-Álvarez
authors.affiliation: †Departamento de Química Física y Analítica, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Arturo J
authors.initials: AJ
authors.lastname: Miranda-Ordieres
authors.affiliation: ‡Departamento de Química, Centro de Ciências da Natureza. Universidade Federal do Piauí, Teresina, 64049-550 PI, Brasil.
authors.forename: J Ribeiro
authors.initials: JR
authors.lastname: Dos Santos Junior
authors.affiliation: §Instituto de Ciências Biológicas, Universidade de Pernambuco, Recife, 50670-901 PE, Brasil.
authors.forename: Rosana A
authors.initials: RA
authors.lastname: da Silva Fonseca
authors.affiliation: †Departamento de Química Física y Analítica, Universidad de Oviedo, 33006 Oviedo, Spain.
authors.forename: Maria Jesús
authors.initials: MJ
authors.lastname: Lobo-Castañón
mesh_terms: D017796:Caulimovirus / Q000235:genetics; D004265:DNA Helicases / Q000378:metabolism*; D017931:DNA Primers / Q000378:metabolism; D004279:DNA, Viral / Q000032:analysis* / Q000378:metabolism; D055664:Electrochemical Techniques; D057230:Limit of Detection; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D009693:Nucleic Acid Hybridization; D030821:Plants, Genetically Modified / Q000235:genetics* / Q000821:virology; D011401:Promoter Regions, Genetic
chemical_list: D017931:DNA Primers; D004279:DNA, Viral; D004265:DNA Helicases