P3-28 Evaluation of a Suite of Loop-mediated Isothermal Amplification Assays for the Rapid, Reliable, and Robust Detection of Shiga Toxin-producing Escherichia coli in Produce

Wednesday, July 31, 2013
Exhibit Hall (Charlotte Convention Center)
Fei Wang, University of Maryland-College Park, College Park, MD
Qianru Yang, Louisiana State University, Baton Rouge, LA
Kelly Jones, U.S. Food and Drug Administration, Laurel, MD
Jianghong Meng, University of Maryland-College Park, College Park, MD
Beilei Ge, U.S. Food and Drug Administration, Laurel, MD
Introduction: Shiga toxin-producing Escherichia coli (STEC) is a leading cause of produce-associated outbreaks in the United States. It is critical to have rapid, reliable, and robust STEC detection methods to better ensure produce safety. A suite of loop-mediated isothermal amplification (LAMP) assays were developed recently for STEC detection. However, they have not been evaluated using an extensive collection of strains or complex produce matrices.

Purpose: The purpose of this study was to further evaluate LAMP performance in comparison with real-time quantitative PCR (qPCR) using a large panel of strains, and to validate the method for the rapid, reliable, and robust detection of STEC in various produce items.

Methods: The specificity of LAMP assay was evaluated with 160 bacterial strains, and the sensitivity was tested with seven STEC strains of adulterant O serogroups in pure culture and spiked produce samples (spinach, lettuce, and sprouts). To simulate real-world contamination events, produce samples were surface-inoculated with low levels (1-10 CFU/25 g) of individual STEC strain, and held at 4°C for 48 h before testing with LAMP and qPCR. Six commonly used DNA extraction procedures were also compared for their effect on assay performance.

Results: All STEC targets and their subtypes were accurately detected. The detection limits of various targets were approximately 2 to 20 cells/reaction in pure culture and 105 to106 CFU/25 g in spiked produce samples, except for stx2c, eaeβ, eaeε1, and eaeγ2/θ. In produce samples spiked with low levels of respective STEC strains, LAMP consistently achieved accurate detection after 6 to 8 h of enrichment, with the exception of sprouts. Different DNA extraction methods also yield varied results.

Significance: The research provided a rapid, reliable, and robust method for detecting STEC in produce samples during routine sampling and analysis. The challenge with sprouts detection by both LAMP and qPCR calls for specific attention and further analysis.