ERP Micro December 2019

sometimes cause kidney failure and even death. This enterohemorrhagic E. coli (EHEC) is a pathogenic E. coli strain that produce Shiga toxins (Stx) and causes hemorrhagic colitis (HC) and the potentially life-threatening hemolytic uremic syndrome (HUS). The HUS infection produces toxic substances that destroy red blood cells, causing kidney injury (1, 2,3). Principle of the Method Assurance GDS for E. coli O157:H7 Tq (GDS EHEC) is an automated gene-based assay that uses proprietary probes and specific primers directed against a highly conserved DNA sequence of the target organism. The GDS EHEC method detects strains of Escherichia coli O157:H7 and toxigenic E . coli O157 NM (Strains of E. coli O157:H7 that have lost the flagellar “H” antigen become nonmotile and are designated “NM”). The assay is designed to be highly selective and does not detect micro-organisms that are potential cross-reactors in antibody-based assays, including E. coli O157 that are not H7 or NM, and other microorganisms that express O157 antigen but are not E. coli O157:H7 (4). The GDS EHEC method also uses a proprietary IMS device, PickPen TM , and associated reagents that concentrate populations of target microorganisms, thereby providing increased sensitivity and elimination of potential competitive microflora. The assay is based on DNA amplification and detection by real-time PCR. The GDS method was adopted AOAC Official Method Final Action in 2007 (5). It was modified in 2013 for testing of composite-sized (375 g) samples (6). The objective of this study is to demonstrate equivalance of a matrix extension and two minor modifications for the GDS EHEC method to the reference methods. First is the extension of matrices to include finely textured beef (FTB, 375 g), carcass cloths and green onions (25 g). The FTB samples will be frozen when added to the media for enrichment. Secondly, a modification is the use of an automated DNA extraction system, Assurance® GDS PickPen TM PIPETMAX® (PPMX), for sample preparation. The PPMX is a modular automated system to complete the routine pipetting and the PickPen TM IMS concentration of target organism. The machine automates all reagent dispensing, performs the PickPen TM IMS protocols and dispenses concentrated DNA into the Amplification tubes. The PPMX will be validated as an alternative sample preparation method alongside the standard method for GDS EHEC for beef trim, FTB and carcass cloths. Finally, the third modification is an alternative amplification tube size for the assay. A smaller alternative tube size for the amplification tube (HT, High Throughput) has previously been developed for GDS EHEC. The internal amplification contents remain unchanged; only the exterior tube size is modified. The HT amplification tube will also be validated alongside the standard (size) amplification tube for GDS EHEC for use by the manual and automated DNA extraction methods by demonstrating consistency of the two tube formats. The HT amplification tubes will be analyzed on beef trim, FTB and carcass cloth.

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