Development of an one-step centrifugal microdevice for real-time and multiplex identification of food poisoning bacteria

Abstract

We demonstrate a centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria from milk samples. The microdevice is equipped with triplicate functional units and each unit has four reaction chambers, thereby making it possible to perform twelve reactions simultaneously. The integrated microdevice consists of two layers: the samples were injected into a sample reservoir of a bottom layer, and the RPA reagents were injected into a top layer. For multiplex bacterial detection, the target specific primers and probes were freeze-dried in each reaction chamber. The injected samples and reagents could be equally aliquoted and dispensed into each reaction chamber by centrifugal force, and then the multiplex direct-RPA reaction was executed. The target genes of bacteria spiked in the milk could be amplified at $39 ^\circ C$ without DNA extraction step by using the direct-RPA cocktails, which were the combination of a direct PCR buffer and RPA enzymes. As the target gene amplification proceeded, the increased fluorescent signals of the reaction chambers were recorded every 2 min for real-time analysis. The entire process, including the sample distribution, the direct-RPA reaction, and the real-time analysis, were accomplished with a custom-made portable genetic analyzer and a miniaturized optical detector. Three kinds of food poisoning bacteria (Salmonella enterica, E.coli 0157:H7, and Vibrio parahae-molyticus) were successfully detected even with a detection sensitivity of single cell within 30 min. Monoplex, duplex, and triplex bacteria detection was successfully conducted with high selectivity. By combining the direct PRA on the integrated microdevice, the proposed miniaturized genetic analysis system can provide an advanced on-site food poisoning bacteria analysis platform with high speed, sensitivity, and multiplicity.