ERP Micro December 2019

be analyzed. Since exclusivity is performed with direct cultural analysis rather than IMS concentration, the PPMX was performed on inclusivity (with IMS method) only. For the inclusivity evaluation of the Assurance® GDS for E . coli O157:H7 assay using PPMX, 50 isolates of E . coli O157:H7 were cultured in mEHEC® media for 18–24 h at 41–43°C. The culture tubes were diluted to 100x the Limit of Detection (LOD 50 ). The target organisms were enriched, serially diluted and tested, consistent with Appendix J with GDS, following the manufacturer’s Directions for Use. These prepared inclusivity cultures were processed by Assurance® GDS PPMX.

AOAC Official Method 2005.04 Escherichia coli O157:H7 in Selected Foods Assurance ® GDS for E . coli O157:H7 Tq

First Action 2005 Final Action 2007

Revised First Action 2011 Revised First Action 2012 Revised First Action 2019

(Applicable to detection of E . coli O157:H7 in raw ground beef, beef trim, finely textured beef, carcass cloths, orange juice, apple juice, fresh vegetables, green onions and sprout process water) E . coli O157:H7 are pathogenic bacteria. Symptoms of infection include: bloody diarrhea and cramping, little to no fever and hemolytic uremic syndrome. Decontaminate all spent media and equipment used in test prior to disposal of media or re-use of equipment. Note : Employ proper environmental and procedural precautions for the handling of reagents and equipment used to perform genetic-based assays. See Table 2005.04 (original study, 2005) for the results of the interlaboratory study supporting acceptance of the method. A. Principle The Assurance ® GDS for E . coli O157:H7 Tq method is a gene-based assay that uses specific primers and proprietary probes directed against a highly conserved DNA sequence in the target organism. The method uses enrichment in modified EHEC (mEHEC®) broth. After enrichment, populations of target microorganisms are concentrated by using a proprietary concentration (PickPen TM ) device and reagents or processed with an automated concentration instrument. The concentrate is then transferred to a conical reaction vessel containing all necessary amplification reagents (Amplification tube). The vessel is sealed and placed in a rotary thermocycler PCR instrument (Rotor-Gene ® ) which uses proprietary software for simultaneous amplification and detection of target DNA. All tests, positive and negative, are indicated at the Caution :

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