Real-time loop-mediated isothermal DNA amplification in compact disc micro-reactors

Autores de IIS La Fe

• Rosa Puchades Pla

  Autor

Participantes ajenos a IIS La Fe

• Santiago-Felipe S
• Tortajada-Genaro LA
• Carrascosa J
• Maquieira Á

Grupos

• Unidad Mixta de Investigación en Nanomedicina y Sensores

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 (2 mm diameter and 1.1 mm 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 mu g/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 mu L of reaction), portability, ease-of-use, and rapid low-cost high-throughput analyses. (C) 2015 Elsevier B.V. All rights reserved.

Keywords

• DNA amplification; LAMP; Isothermal real-time; Micro-reactors; Compact disc