Point-of-care genetic analysis for multiplex pathogenic bacteria on a fully integrated centrifugal microdevice with a large-volume sample.

Abstract

We present a fully integrated portable centrifugal microsystem for multiplex detection of food poisoning bacteria with a large volume of sample up to 1\u202fmL. The microsystem consists of a portable genetic analyzer and a fully integrated centrifugal microdevice. The centrifugal microdevice is designed with two units: a 3D printed solution-loading cartridge and a centrifugal microfluidic disc. All the essential solutions for loop-mediated isothermal amplification (LAMP) reaction are stored inside the cartridge, and orderly released into centrifugal microdevice by a rotation program. Each unit of the device is designed with 20 reaction chambers for simultaneous detection of food-borne bacteria in one test. To increase the amount of a sample to 1\u202fmL, we incorporated the super absorbent polymer (SAP) in the waste chamber to absorb the sample and the washing solution during the device operation. The whole process was automatically conducted including designated solution release, bead-based DNA extraction, isothermal gene amplification by Eriochrome Black (EBT)-mediated LAMP reaction, and colorimetric and UV-visible detection of amplicons. The ratio between Abs640nm and Abs570nm was used as a criterion to confirm the positive result, and the result was positive upon the condition of Abs640/Abs570 ≥ 1.0. To demonstrate the pathogenic bacteria detection on our proposed microsystem, we targeted three kinds of bacteria (Escherichia coli O157:H7, Salmonella typhimurium, and Vibrio parahaemolyticus) for monoplex and multiplex detection. The whole process from sample to result was completed within 1\u202fh with a low limit of detection (LOD) of 102\u202fcells/mL.