A self-contained microfluidic in-gel loop-mediated isothermal amplification for multiplexed pathogen detection

In this work, we propose a microfluidic in-gel loop-mediated isothermal amplification (gLAMP) containing preloaded reagents as a self-contained microdevice for simultaneous detection of multiple pathogenic bacterial DNA in near point of care settings. The microchip consists of multiple channels with reaction chambers accommodating low-melting-point agarose and all LAMP reagents except the DNA samples, which can be kept at 4 degrees C for long-term storage. The current microchip enables simultaneous tests of four different bacterial DNA targets. After DNA samples were introduced into the microchip, gLAMP reactions were carried out on a hot plate at 65 degrees C. The gLAMP reaction conditions including the concentrations of the fluorescence indicator, Bst DNA polymerase, primers, and Mg2+ ions were further optimized. Under the optimized conditions, detection of foodborne bacteria in serum samples, including Escherichia coli, Proteus hauseri, Vibrio parahaemolyticus, and Salmonella subsp. Enterica were demonstrated with high selectivity and sensitivity (as low as 3 copies/mu L). The gLAMP microchip provides a simple and easy-to-operate platform for LAMP-based pathogen detection, which holds high potential for future applications in point-of-care settings. (C) 2016 Elsevier B.V. All rights reserved.