Simultaneous Detection of Five Foodborne Pathogens Using a Mini Automatic Nucleic Acid Extractor Combined with Recombinase Polymerase Amplification and Lateral Flow Immunoassay

Abstract

In recent years, foodborne disease outbreaks have caused huge losses to the economy and have had severe impacts on public health. The accuracy and variety of detection techniques is crucial to controlling the outbreak and spread of foodborne diseases. The need for instruments increases the difficulty of field detection, while manually-handled samples are subject to user error and subjective interpretation. Here, we use a mini automatic nucleic acid extractor combined with recombinant polymerase amplification (RPA) and lateral flow immunoassay (LFIA) for simultaneous quantitative detection of five major foodborne pathogens. The pre-treatment device using the magnetic bead method allows for nucleic acid extraction of the reagent tank without manual operation, which is highly efficient and stable for preventing aerosol contamination. The nuc gene of Staphylococcus aureus, the toxR gene of Vibrio parahaemolyticus, the rfbE gene of Escherichia coli O157:H7, the hlyA gene of Listeria monocytogenes, and the fimY gene of Salmonella enterica were used as target fragments. The labeled antibody concentration is optimized on the LFIA to find the equilibrium point for the binding capacity of the five chemical markers and to efficiently and accurately visualize the bands. The RPA assay shows an optimal performance at 37 °C for 15 min. The optimized RPA-LFIA detection limit can reach 101 CFU/mL. There was no cross-reactivity among forty-eight strains. Furthermore, the average recoveries in spiked food samples were 90.5–104.5%. In summary, the RPA-LFIA established in this study can detect five pathogenic bacteria simultaneously with little dependence on laboratory equipment, and it has promising prospects for screening in low-resource areas.

Rapid detection of multiple resistance genes to last-resort antibiotics in Enterobacteriaceae pathogens by recombinase polymerase amplification combined with lateral flow dipstick

“…Thus, in our study, a fast, innovative boil-up method for crude DNA extraction (taking around 10 min for the entire extraction process) from Enterobacteriaceae pathogens was used to increase the time savings of the multiplex RPA procedure. Studies have confirmed the application of multiplex RPA assays for simultaneously detecting multiple foodborne pathogens (Ma et al, 2020;Jin et al, 2022). However, studies on its detection for ARG remain less.…”

“…LFD strips were prepared via methods described in previous studies with some modifications ( Ma et al, 2020 ; Jin et al, 2022 ). Briefly, the LFD strips were composed of continuous superposition: sample pad, conjugate pad with gold nanoparticles (AuNPs), nitrocellulose filter (NC) membrane, adsorption pad as well as backing card.…”

Lateral Flow Recombinase Polymerase Amplification Assays for the Detection of Human Plasmodium Species

“…Jin et al reported a multiplex RPA assay for simultaneous detection of five foodborne pathogens with 10 CFU/mL detection limit. 33 Larrea-Sarmiento et al reported a multiplex RPA in the detection of agricultural important bacteria (genus Clavibacter and C. nebraskensis) and achieved a detection limit of 3,000 copies and 30 copies for Clavibacter-and C. nebraskensis primers/probes, respectively. 34 Garrido-Maestu et al showed a limit of detection below 10 CFU/25 g in detection of Listeria monocytogenes in dairy products by using real-time multiplex RPA.…”

Multiplex detection of the big five carbapenemase genes using solid-phase recombinase polymerase amplification

“…For example, RPA NALFIA (nucleic acid lateral flow immunoassay) approaches discussed in the literature require multiple antibodies and probes to generate lateral flow sandwich immunoassays. 42,43…”