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

• Published in: Biosensors & bioelectronics Vol. 79; pp. 300 - 306
• Main Authors: Santiago-Felipe, Sara, Tortajada-Genaro, Luis A., Carrascosa, Javier, Puchades, Rosa, Maquieira, Ángel
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 15-05-2016
• Subjects: Amplification; Animals; Bacteria; Biosensing Techniques; Cattle; Colorimetry; Compact Disks; Deoxyribonucleic acid; Devices; Disc drives; DNA - genetics; DNA - isolation & purification; DNA amplification; Food Microbiology; Isothermal real-time; LAMP; Meat - microbiology; Micro-reactors; Nucleic Acid Amplification Techniques; Real time; Salmonella; Salmonella - isolation & purification; Salmonella - pathogenicity; Micro-reactors; DNA amplification; LAMP; Isothermal real-time; Compact disc
• ISSN: 0956-5663; 1873-4235
• DOI: 10.1016/j.bios.2015.12.045

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 15min, with high selectivity and sensitivity (5CFU/mL and 10µg/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 (3µL of reaction), portability, ease-of-use, and rapid low-cost high-throughput analyses.
•Isothermal non fluorescence device for low-cost portable DNA quantification.•LAMP performed on micro-reactors embedded onto compact discs.•Real-time curves measured by cyclic scanning using the optics of a DVD drive.•Assay evaluated for pathogen determination and meat identification.