Improved Real-Time PCR Detection of Listeria in Environmental Samples after Rapid Single Step Enrichment

Introduction: Finished product testing for Listeria monocytogenes often provides limited information on the safety status of a food by neglecting recontamination risks during slicing and packaging steps and because in real world contamination events the individual portion prevalence rate in a production lot can be low. Implementation of an effective environmental monitoring program is becoming an increasingly relevant proactive food safety strategy for controlling L. monocytogenes in food processing facilities.

Purpose: The goal of this study was to evaluate a sensitive and rapid method for monitoring L. monocytogenes and Listeria genus in environmental samples using a rapid, single-stage enrichment in Actero Listeria Enrichment Media followed by processing with the BAX System real-time PCR assays for Listeria and L. monocytogenes.

Methods: Environmental samples were collected from food contact (stainless steel and plastic) and non-food contact (ceramic, sealed concrete and rubber) surfaces. Stressed overnight by desiccation, five different Listeria species (L. monocytogenes, L. innocua, L. ivanovii, L. seeligeri and L. welshimeri) were used alone or together with high numbers competitive background flora (E. faecalis or P. aeruginosa) to artificially inoculate surfaces. Samples were enriched in Actero Listeria Enrichment Media, then processed with the DuPont BAX System real-time PCR assays.

Results: A total of 930 environmental sponge samples were evaluated in two large-scale, unpaired validation studies in comparison with the USDA-FSIS and Health Canada reference methods, including 570 samples analysed by certified independent laboratories. Enrichment phase optimization studies allowed for reduction by up to 20 hours the time required to detect Listeria. The method comparison studies demonstrated no false positive results and a false negative rate of 0.3%. According to the Probability of Detection statistical model, the candidate method showed equivalent or superior performance to the reference methods. 

Significance: Thus, a reliable and rapid alternative method has been developed and validated that can be successfully used as a monitoring tool for controlling Listeria in the food industry.