Development of a high-throughput centrifugal loop-mediated isothermal amplification microdevice for multiplex foodborne pathogenic bacteria detection

Abstract Since the foodborne pathogenic bacteria cause serious diseases and socio-economic losses throughout the world, the molecular diagnosis of foodborne poisoning pathogen in the early stage is essential for preventing excessive damages. In this study, a colorimetric based foodborne pathogen detection on the centrifugal microdevice was demonstrated in a high throughput manner. The developed centrifugal microdevice consists of a sample reservoir, a spiral shaped sample injection microchannel, 24 aliqouting chambers (2.5 μL each), cross capillary valves, and 24 reaction chambers on a single device. Once the sample was loaded, the rotation speed at 850 rpm allowed the sample solution to be divided into 24 aliqouting chambers. Then, the samples loaded in the aliqouting chambers could be injected into the reaction chambers at 5000 rpm evenly. The centrifugal device was placed in an oven at 66 °C for target gene amplification. As a gene amplification method, loop mediated isothermal amplification (LAMP) was used in which specific primer sets targeting three kinds of foodborne pathogens (Escherichia coli O157:H7, Salmonella typhimurium, and Vibrio parahaemolyticus) were included. The presence of each pathogen was identified by Eriochrome Black T (EBT)-mediated colorimetric change from purple to sky blue with the naked eye, and the color of each reaction chamber was analyzed by a ratiometric image processing method. Green/Red (G/R) and Blue/Red (B/R) ratios were set as the criteria to differentiate negative results from positive ones. The threshold values for G/R (0.915