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

Sarah Azinheiro,Joana Carvalho,Marta Prado,Alejandro Garrido-Maestu

doi:10.3390/foods9091249

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.

Highlights

With a mortality rate of 15.6%, reported by EFSA and ECDC in 2018, listeriosis is one of the most serious food-borne diseases under EU surveillance, and since year 2000 an increase in the number of cases was observed [1]
We aimed to develop a novel method combining Recombinase Polymerase Amplification (RPA) amplification with lateral flow (LF) naked-eye detection, for the rapid assessment of L. monocytogenes contamination on food processing surfaces
To determine the best primer set to be used with the RPA in combination with LF detection, first the lowest concentration of a L. monocytogenes DNA with each set, yielding a positive result was determined; and second, the concentration of L. monocytogenes cells needed to have a positive result by RPA-LF was determined, this was performed by preparing ten-fold dilutions from an ON

Summary

Introduction

With a mortality rate of 15.6%, reported by EFSA and ECDC in 2018, listeriosis is one of the most serious food-borne diseases under EU surveillance, and since year 2000 an increase in the number of cases was observed [1]. Due to the severity of the disease, L. monocytogenes contamination needs to be accurately controlled. Aseptic measures are needed to avoid cross-contaminations in the processing environment [4]. Due to its ability to survive in harsh environments, such as low temperature [5], and high salt concentrations, along with its capacity to create biofilms [6], L. monocytogenes can persist in the food supply chain, even with regular sanitation, which may lead to contamination of food [7]. Processing plants should be tested for the presence of this pathogen to identify possible problems in the food factory and to implement corrective measures [8]

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