A centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria

Abstract

In this study, we developed a centrifugal direct recombinase polymerase amplification (direct-RPA) microdevice for multiplex and real-time identification of food poisoning bacteria contaminated milk samples. The microdevice was designed to contain identical triplicate functional units and each unit has four reaction chambers, thereby making it possible to perform twelve direct-RPA reactions simultaneously. The integrated microdevice consisted of two layers: RPA reagents were injected in the top layer, while spiked milk samples with food poisoning bacteria were loaded into sample reservoirs in the bottom layer. For multiplex bacterial detection, the target gene-specific primers and probes were dried in each reaction chamber. The introduced 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 milk could be amplified at 39 °C without a DNA extraction step by using the direct-RPA cocktails, which were a combination of a direct PCR buffer and RPA enzymes. As the target gene amplification proceeded, the increased fluorescence signals coming from the reaction chambers were recorded in real-time at an interval of 2 min. The entire process, including the sample distribution, the direct-RPA reaction, and the real-time analysis, was accomplished with a custom-made portable genetic analyzer and a miniaturized optical detector. Monoplex, duplex, and triplex food poisoning bacteria (Salmonella enterica, Escherichia coli O157:H7, and Vibrio parahaemolyticus) detection was successfully performed with a detection sensitivity of 4 cells per 3.2 μL of milk samples within 30 min. By implementing the direct-PRA on the miniaturized centrifugal microsystem, the on-site food poisoning bacteria analysis would be feasible with high speed, sensitivity, and multiplicity.

Recombinase polymerase amplification: Basics, applications and recent advances

“…Another approach successfully implemented for the analysis of diluted crude DNA extracts from blood or swab samples consisted of heating the sample with AVL buffer and Trizol, followed by centrifugation [46]. RPA can also be carried out directly in urine [47], pleural fluids [48], seed powders [10], milk [49] and stool samples [50], only requiring heat lysis, direct lysis with nuclease free water or use of the EzWay™ Direct PCR buffer [49]. However, another study found that while 1,25% (v/v) of urine has no impact on amplification efficacy, 10% (v/v) did inhibit amplification when small amounts of target DNA were present in the sample (100 fg), but, this inhibition is not observed when the target DNA concentration is higher (10 pg), even at 10% (v/v) urine [51].…”

“…Other reports detail pseudomultiplexing platforms through parallelised single reactions, using foil based centrifugal microfluidic cartridges with stored reagents [49,58], digital versatile discs (DVD) [41,42,44], vacuum degassed microfluidic cartridges [59] or polylactic acid/polycarbonate chips [6].…”

An improved recombinase polymerase amplification assay for visual detection of Vibrio parahaemolyticus with lateral flow strips

“…Given the long food supply chains and extensive mariculture industry developments of this era, the detection technologies are required to be simple and convenient for on‐site environment and resource‐limited regions. Application of RPA technology to V. parahaemolyticus detection has led to the development of several assays with added convenience as compared to the PCR‐based methods (Choi et al., 2016; Geng et al., 2019; Yang et al., 2018). Combining the LFS test with RPA can further simplify the detection and provide real instrument‐free assays.…”

“…The end‐point detection of RPA products can be carried out with Agarose gel electrophoresis, fluorescent reading with nucleic acid dyes, spectrophotometry, or lateral flow strip (LFS) assays (Piepenburg, Williams, Stemple, & Armes, 2006). Using the RPA technology, several detection assays have been developed for V. parahaemolyticus (Choi et al., 2016; Geng et al., 2019; Yang et al., 2018). These assays have eliminated the use of thermal cyclers and added convenience for on‐site detection.…”

<p>Current and Future Perspectives on Isothermal Nucleic Acid Amplification Technologies for Diagnosing Infections</p>

“…These include digital RPA known as the slipchip 96,97 and microchip; 98 multiplex RPA for bacteria namely Salmonella spp and Chronobacter spp, 99 Listeria monocytogenes and Salmonella enterica serotype Enteritidis, 100 Group B streptococci; 101 parasites (Giardia, Cryptosporidium, and Entamoeba) 102 and fungus (Botrytis cinerea, Pseudomonas syringae, and Fusarium oxysporum); 103 mobile RPA in a suitcase which was named diagnostics-in-a-suitcase (Dias); 104,105 microfluidic integrated RPA; [106][107][108][109] and the one-step RPA assay. 110 While a slip chip is microfluidic device lacking pumps and valves, a microchip contains pumps and valves that aid movement of sample and reaction reagents through channels linking reaction chambers. Although the initial patent (EP 0481065 B) over RPA has expired, patent rights still exist for some of the improvements it has experienced.…”