Development of a Rapid Enteric Pathogen Indicator for Leafy Greens
Erica Miller 1, Nicole Gasdik 2, Rafael Davila 2, Alexa Grace Baldwin 3, and Christopher Crowe 2
Eurofins Microbiology Laboratories, Inc.: 1 Louisville, KY; 2 Salinas, CA; 3 Pathotrak, College Park, MD
ABSTRACT
Introduction
Salmonella and STEC contamination is a major concern in fresh leafy green products. Testing for pathogens and indicator organisms such as coliforms and E. coli can take up to 24 hours or more to return results. Methods with faster turnaround times are sorely needed for produce, which has short shelf life
and very tight supply chain timelines.
Purpose
To develop a molecular method that detects indicators of enteric pathogen contamination and requires less than six hours of enrichment.
Methods
Samples of 20 different leafy green and other fresh produce matrices were artificially inoculated (7 test portions, each of 375 g per matrix; 133 portions in total) with < 30 CFU of Salmonella and/or STEC and enriched in pre-warmed buffered peptone water containing 0.5% polysorbate-20. Samples were incubated at 42°C for 5.5 hours, and then the entirety of each enrichment was concentrated using the PathotrakTM concentration system. Concentrated samples were tested using the Enteric Pathogen Risk Indicator (EPRI) real-time PCR kit (Gold Standard Diagnostics) in parallel with being tested for both Salmonella and STEC by real-time PCR.
Results
For all 20 matrices tested, all inoculated replicates produced a positive detection using EPRI real-time PCR following 5.5 hours of enrichment and sample concentration. The presence of Salmonella and/or STEC was confirmed by pathogen-specific PCR in each sample. Thus, every matrix passed the FDA matrix extension protocol requiring detection in 7/7 inoculated samples. There was no observed inhibition from the produce or concentration procedures in the PCR reaction.
Significance
These data demonstrate the feasibility of a rapid protocol to assess the potential presence of enteric pathogens in fresh produce matrices. If the EPRI assay indicates that enteric pathogens may be present, this data demonstrates that further testing of the concentrated sample for Salmonella and/or STEC is also possible.
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