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

Authors：Santiago Felipe Sara; Tortajada Genaro Luis A; Carrascosa Javier; Puchades Rosa; Maquieira Angel^*

Source：Biosensors and Bioelectronics, 2016, 79: 300-306.

DOI：10.1016/j.bios.2015.12.045

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

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.