Application of Recombinase Polymerase Amplification with Lateral Flow for a Naked-Eye Detection of Listeria monocytogenes on Food Processing Surfaces

Abstract

The continuous contamination of foods with L. monocytogenes, highlights the need for additional controls in the food industry. The verification of food processing plants is key to avoid cross-contaminations, and to assure the safety of the food products. In this study, a new methodology for the detection of L. monocytogenes on food contact surfaces was developed and evaluated. It combines Recombinase Polymerase Amplification (RPA) with the lateral flow (LF) naked-eye detection. Different approaches for the recovery of the bacteria from the surface, the enrichment step and downstream analysis by RPA-LF were tested and optimized. The results were compared with a standard culture-based technique and qPCR analysis. Sampling procedure with sponges was more efficient for the recovery of the bacteria than a regular swab. A 24 h enrichment in ONE broth was needed for the most sensitive detection of the pathogen. By RPA-LF, it was possible to detect 1.1 pg/µL of pure L. monocytogenes DNA, and the complete methodology reached a LoD50 of 4.2 CFU/cm2 and LoD95 of 18.2 CFU/cm2. These results are comparable with the culture-based methodology and qPCR. The developed approach allows for a next-day detection without complex equipment and a naked-eye visualization of the results.

State of the art: Lateral flow assays toward the point‐of‐care foodborne pathogenic bacteria detection in food samples

“…The advanced technique has enabled for a next‐day determination without sophisticated apparatus and a naked‐eye imagining of the outcomes. It has been represented that the achieved outcomes have been comparable with those of qPCR and culture‐based methods (Azinheiro et al., 2020).…”

Naked‐eye detection strategies coupled with isothermal nucleic acid amplification techniques for the detection of human pathogens

“…(c) Diagram (top row) and real samples (bottom row) of LAMP reactions combined with AuNP functionalized with oligonucleotide probes (image adapted from Arunrut et al., 2016). (d) Schematic representation of a LF strip (image adapted from Koczula & Gallotta, 2016) and real strips targeting L. monocytogenes (image from Azinheiro et al., 2020)…”

“…Due to their simplicity of use and low cost, these type of assays have been combined with different types of isothermal methods such as LAMP, RPA, or SEA for naked‐eye detection of different microorganisms in foods, surfaces, and even clinical samples (Azinheiro et al., 2020; Du et al., 2018; Wang et al., 2020; Zasada et al., 2020), with particular interest in low‐resource settings due to the need of very little instrumentation (Mei et al., 2019; Poulton & Webster, 2018; Srisrattakarn et al., 2020; van Dam et al., 2013).…”

“…(2006). It needs a pair of primers that are recommended to be slightly longer than those of PCR (≈30 bp), even though PCR primers are also suitable (Azinheiro et al., 2020). RPA employs recombinase–primer complexes to scan double‐stranded DNA and facilitate strand exchange at cognate sites.…”

A Rapid and Sensitive Europium Nanoparticle-Based Lateral Flow Immunoassay Combined with Recombinase Polymerase Amplification for Simultaneous Detection of Three Food-Borne Pathogens

“…Listeria monocytogenes is a rod-shaped, Gram-positive food-borne bacterium with high tolerance to different growth conditions, including high concentrations of salt and low pH levels [ 5 ]. Due to its ability to survive in harsh environments, it can continue to exist in the food supply chain even under conventional sanitary conditions, which may lead to food contamination [ 6 ]. Vibrio parahaemolyticus is a curved rod-shaped, Gram-negative halophilic bacterium that is mainly distributed in various seafood products, such as fish, shrimp, and shellfish [ 7 , 8 , 9 ].…”