Toward a rapid and reliable pathogen detection system in produce.

Toward a rapid and reliable pathogen detection system in produce.

Jan. 1, 2012 - Dec. 31, 2013

2012-168

$152,591.00

Jianghong Meng, Ph.D.
University of Maryland

Beilei Ge, Ph.D.

Final Report

Recent outbreaks linked to Salmonella- and E. coli-contaminated produce pose a significant threat to public health and the produce industry. Through a project previously funded by CPS, we developed a rapid, accurate, yet simple and cost-effective molecular testing method termed “LAMP” for live Salmonella detection in produce. In this proposal, LAMP will be further developed and validated to be a rapid andreliable pathogen detection system for both Salmonella and E. coli in produce. Specifically, we will develop and validate LAMP detection of low levels of these pathogens in various produce items using conditions mimicking real-world contamination events. Specific objectives include: 1) To develop and evaluate LAMP assays for E. coli pathogens; 2) To evaluate the robustness of the LAMP detection system for both Salmonella and E. coli; and 3) To validate the system in complex produce matrices. Rapid, accurate, simple, and robust detection of important human pathogens in produce will provide the produce industry an invaluable tool to better control potential microbial contaminants, therefore significantly reducing the number of outbreaks and illnesses associated with fresh produce.

Technical Abstract

Salmonella, Escherichia coli O157:H7, and more recently non-O157 Shiga toxin-producing E. coli (STEC) are major human pathogens causing food safety concerns. Recent outbreaks linked to produce contaminated with these pathogens greatly undermined consumer confidence and heightened the need for applied targeted research from field to fork. Among multifaceted approaches to better ensure produce safety and prevent future outbreaks, rapid, accurate, simple, cost-effective, and reliable detection methods are especially needed in order to promptly identify pathogen contamination problems throughout the production, processing, and distribution of produce. However, such methods are currently lacking and when compared to method development in meat and poultry, pathogen detection in produce is still in its infancy. With funds from CPS (project 2008-11), we carried out a project using propidium monoazide (PMA) coupled with a novel DNA amplification technique, loop-mediated isothermal amplification (LAMP) to detect live Salmonella cells in produce (an article to appear in the June 2011 issue of the journal Applied and Environmental Microbiology). Although reported to be rapid, accurate, quantitative, and highly specific for viable Salmonella cells, the LAMP assay has not been validated using complex produce matrices mimicking real-world contamination events. Additionally, although several LAMP assays for Shiga toxin genes and STEC O157 have been described, currently there are no LAMP assays addressing non-O157 STEC detection. The overarching goal of the proposed project is to develop a rapid and reliable LAMP detection system for Salmonella, STEC O157, and non-O157 STEC, and validate the system with produce items (cantaloupe, lettuce, pepper, spinach, sprout, and tomato) surface-contaminated with low levels of these pathogens. Firstly, a set of LAMP assays for major non-O157 STEC serogroups (O26, O45, O103, O111, O121 and O145) will be developed. Secondly, the robustness of the LAMP system for both Salmonella and STEC detection will be evaluated using abusive temperature, pH, and the addition of soil, chlorophyll, and produce enrichment broth. Finally, using surface-inoculated produce samples, the efficacies of this LAMP system in detecting low levels of these pathogens will be validated. Upon completion, this project will generate a rapid and reliable LAMP detection system readily applicable to the produce industry for the rapid, accurate, simple, and robust detection of pathogens that may potentially contaminate raw and finished products, so that timely intervention strategies may be implemented. This improvement will provide significant benefits to produce growers, harvesters, processors, distributors, retailers, and the ultimate consumers by better ensuring produce safety, therefore significantly reducing the risks and illnesses associated with the consumption of fresh produce.