title: Isothermal DNA amplification strategies for duplex microorganism detection.
abstract: A valid solution for micro-analytical systems is the selection of a compatible amplification reaction with a simple, highly-integrated efficient design that allows the detection of multiple genomic targets. Two approaches under isothermal conditions are presented: recombinase polymerase amplification (RPA) and multiple displacement amplification (MDA). Both methods were applied to a duplex assay specific for Salmonella spp. and Cronobacter spp., with excellent amplification yields (0.2-8.6 · 10(8) fold). The proposed approaches were successfully compared to conventional PCR and tested for the milk sample analysis as a microarray format on a compact disc (support and driver). Satisfactory results were obtained in terms of resistance to inhibition, selectivity, sensitivity (10(1)-10(2)CFU/mL) and reproducibility (below 12.5%). The methods studied are efficient and cost-effective, with a high potential to automate microorganisms detection by integrated analytical systems working at a constant low temperature.
authors.affiliation: Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM) - Departamento de Química, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Sara
authors.initials: S
authors.lastname: Santiago-Felipe
authors.affiliation: Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM) - Departamento de Química, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Luis Antonio
authors.initials: LA
authors.lastname: Tortajada-Genaro
authors.affiliation: Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM) - Departamento de Química, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Sergi
authors.initials: S
authors.lastname: Morais
authors.affiliation: Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM) - Departamento de Química, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain.
authors.forename: Rosa
authors.initials: R
authors.lastname: Puchades
authors.affiliation: Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM) - Departamento de Química, Universidad Politécnica de Valencia, Camino de Vera s/n, 46022 Valencia, Spain. Electronic address: amaquieira@qim.upv.es.
authors.forename: Ángel
authors.initials: Á
authors.lastname: Maquieira
mesh_terms: D000818:Animals; D002417:Cattle; D059126:Cronobacter / Q000235:genetics / Q000302:isolation & purification*; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D005516:Food Microbiology / Q000379:methods; D008892:Milk / Q000382:microbiology*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012475:Salmonella / Q000235:genetics / Q000302:isolation & purification*; D046888:Tissue Array Analysis / Q000379:methods*
chemical_list: D004269:DNA, Bacterial
keywords: Isothermal DNA amplification; Microarraying; Milk; Pathogens