title: Real-time loop-mediated isothermal DNA amplification in compact disc micro-reactors.
abstract: An integrated device composed of micro-reactors embedded onto compact discs is proposed for real-time targeted DNA determination. The method principle is based on in-disc loop-mediated isothermal amplification (iD-LAMP) and quantitative optical read-out by a disc drive. In the presence of a target, the turbidimetric or colorimetric properties of reaction solution change, and the transmitted intensity of the disc drive laser modifies according to reaction yield. Monitoring real-time curves allowed the quantitative determination of DNA template amounts. The best amplification/detection results were obtained with micro-reactors (2mm diameter and 1.1mm in depth) drilled on a digital video disc (DVD) and detection based on the colorimetric mode. As proof-of-concept, the assay was applied to detect pathogenic bacteria Salmonella spp. and to identify bovine meat in food samples. Ninety-six samples were simultaneously analysed in 15 min, with high selectivity and sensitivity (5 CFU/mL and 10 µg/g for bacteria and meat, respectively). The in-disc results were comparable to those obtained by conventional LAMP or qPCR approaches. The developed device allows low sample and reagent consumption (3 µL of reaction), portability, ease-of-use, and rapid low-cost high-throughput analyses.
authors.affiliation: Instituto interuniversitario de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, E46071 Valencia, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Santiago-Felipe
authors.affiliation: Instituto interuniversitario de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, E46071 Valencia, Spain.
authors.forename: Luis A
authors.initials: LA
authors.lastname: Tortajada-Genaro
authors.affiliation: Instituto interuniversitario de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, E46071 Valencia, Spain.
authors.forename: Javier
authors.initials: J
authors.lastname: Carrascosa
authors.affiliation: Instituto interuniversitario de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, E46071 Valencia, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Puchades
authors.affiliation: Instituto interuniversitario de Reconocimiento Molecular y Desarrollo Tecnológico (IDM), Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, E46071 Valencia, Spain. Electronic address: amaquieira@qim.upv.es.
authors.forename: Ángel
authors.initials: Á
authors.lastname: Maquieira
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D002417:Cattle; D003124:Colorimetry; D016250:Compact Disks; D004247:DNA / Q000235:genetics / Q000302:isolation & purification*; D005516:Food Microbiology; D008460:Meat / Q000382:microbiology; D021141:Nucleic Acid Amplification Techniques; D012475:Salmonella / Q000302:isolation & purification* / Q000472:pathogenicity
chemical_list: D004247:DNA
keywords: Compact disc; DNA amplification; Isothermal real-time; LAMP; Micro-reactors