A rapid and eco-friendly isothermal amplification microdevice for multiplex detection of foodborne pathogens

Abstract

In this study, a plastic microdevice based on loop-mediated isothermal amplification (LAMP) was fabricated for the amplification and on-chip fluorescence detection of multiple pathogens. Papers infused with LAMP reagents and specific primers were embedded inside the multiple reaction chambers of the microdevice. A solution containing the target pathogens was injected into the sample chamber, located in the center of the microdevice, and evenly distributed to the reaction chambers simultaneously via centrifugal force. For detection, fisetin, a plant-derived fluorophore, was used as the DNA-intercalating dye. Purified DNAs of Escherichia coli O157:H7 (E. coli O157:H7), Salmonella spp., Staphylococcus aureus (S. aureus), and Cochlodinium polykrikoides were successfully amplified and directly detected on the microdevice, where as low as 0.13 and 0.12 ng μL⁻¹ of the DNA of E. coli O157:H7 and S. aureus, respectively, were identified. In addition, the potential of this microdevice for point-of-care testing was further examined by incorporating on-chip sample purification module and testing using a real sample – milk spiked with Salmonella spp. The thermally lysed milk sample was filtered using polydopamine-coated paper embedded inside a sample chamber and seamlessly transported into the reaction chambers by centrifugal force for subsequent LAMP followed by direct on-chip detection inside the reaction chambers in which fisetin-soaked papers were embedded. The limit of detection for Salmonella spp. was determined to be approximately 1.7 × 10² CFU mL⁻¹ using the microdevice. This microdevice is safe, easy to use, selective, and sensitive enough for point-of-care testing to identify foodborne pathogens as well as environmentally harmful microorganisms.