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Novel Assay for Detecting Multiple Toxin-Producing E.coli in meat for new USDA Standards

The current invention from the University of Missouri involves the development of a novel assay to detect multiple Shiga toxin-producing Escherichia coli (STEC), primarily in meat. Specifically, the assay is a two-step multiplex melt-curve real time PCR test for detecting seven STEC serotypes. The assay includes an internal amplification control that ensures robustness, minimizing false negative results. Specific primers are designed and uniquely optimized to detect each individual STEC serotype without cross-reactivity to another STEC serotype. Unlike other methods, this assay does not rely on specific fluorescent probes to detect and discriminate among the target STEC strains, but on unique melting temperatures of specific DNA sequences from each strain, increasing the scope for economic savings and extended test reagent shelf life. The assay has been tested on a multitude of food products such as ground beef, beef trimmings, juices, produce, and poultry products. With a shortened enrichment period and highly sensitive detection level, the assay is sensitive, selective, rapid (10 hours or less), and simple to perform.

There is a current lack of rapid, accurate and sensitive methods for detecting STEC strains that cause serious illnesses, including a fatal hemolytic uremic syndrome (kidney failure). Moreover, molecular based methods that are commercially available are assays that use expensive fluorescent probes or antibody-based approaches and materials with shorter storage lives. In addition to being compatible with other known commercially available techniques, this current assay utilizes convenient and readily accessible materials, making it both cost and time efficient.

The assay has the potential to become a standard assay for STEC detection in the food industry and food regulatory agencies, such as in meat, produce and juice testing.

The assay is sensitive, selective, simple to perform and rapid, taking only 8 to 10 hours to complete
This method complies with new USDA guidelines for non-O157 STEC detection in non-intact beef products established in 2012
The economic savings and regent shelf life are relatively increased because the process is not dependent on fluorescent probes for target detection

Validated for multiple strains of O157 and non O157 STEC on grocery store meat, produce, and juices

Patent Application Filed

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University of Missouri - Columbia

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Pending Patent

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