Recombinase polymerase and enzyme-linked immunosorbent assay as a DNA amplification-detection strategy for food analysis.
Polymerase chain reaction in conjunction with enzyme-linked immunosorbent assay (PCR-ELISA) is a well-established technique that provides a suitable rapid, sensitive, and selective method for a broad range of applications. However, the need for precise rapid temperature cycling of PCR is an important drawback that can be overcome by employing isothermal amplification reactions such as recombinase polymerase amplification (RPA). The RPA-ELISA combination is proposed for amplification at a low, constant temperature (40°C) in a short time (40 min), for the hybridisation of labelled products to specific 5'-biotinylated probes/streptavidin in coated microtiter plates at room temperature, and for detection by colorimetric immunoassay. RPA-ELISA was applied to screen common safety threats in foodstuffs, such as allergens (hazelnut, peanut, soybean, tomato, and maize), genetically modified organisms (P35S and TNOS), pathogenic bacteria (Salmonella sp. and Cronobacter sp.), and fungi (Fusarium sp.). Satisfactory sensitivity and reproducibility results were achieved for all the targets. The RPA-ELISA technique does away with thermocycling and provides a suitable sensitive, specific, and cost-effective method for routine applications, and proves particularly useful for resource-limited settings.
Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM)-Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain.
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Santiago-Felipe
Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM)-Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain.
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Tortajada-Genaro
Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM)-Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain.
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Puchades
Centro de Reconocimiento Molecular y Desarrollo Tecnológico (IDM)-Departamento de Química, Universitat Politècnica de València, Camino de Vera s/n, 46022 Valencia, Spain. Electronic address: amaquieira@qim.upv.es.
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Maquieira
D000485:Allergens / Q000032:analysis; D059126:Cronobacter / Q000235:genetics; D004247:DNA / Q000032:analysis*; D004269:DNA, Bacterial / Q000032:analysis; D004271:DNA, Fungal / Q000032:analysis; D018744:DNA, Plant / Q000032:analysis; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism*; D004797:Enzyme-Linked Immunosorbent Assay; D005504:Food Analysis / Q000379:methods*; D005516:Food Microbiology; D005670:Fusarium / Q000235:genetics; D021141:Nucleic Acid Amplification Techniques; D010944:Plants / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics; D045522:Recombinases / Q000378:metabolism*; D012475:Salmonella / Q000235:genetics; D013696:Temperature
D000485:Allergens; D004269:DNA, Bacterial; D004271:DNA, Fungal; D018744:DNA, Plant; D045522:Recombinases; D004247:DNA; D004259:DNA-Directed DNA Polymerase
Allergen; ELISA; Food safety; GMO; Isothermal amplification; Pathogen