ERP Micro December 2019

0 cfu Low High

5

E. coli O157:H7 (ATCC 43890)

MicroTally TM Cloth

Carcass cloth

280 sq. inch

MLG 5C.00

20

5

1 ATCC – American Type Culture Collection

Test Portion Analysis by GDS EHEC Fresh raw beef, frozen FTB (375 g test portions), green onions and carcass cloths were enriched and incubated according to the GDS EHEC instructions for use. A single set of thirty test portions was enriched following an unpaired study design. For the GDS method, 25 g samples were enriched with 225 mL of mEHEC ® broth; 375 g samples were enriched with 1500 mL of mEHEC® broth. Individual carcass cloths were enriched with 200 mL of mEHEC® broth. Samples were macerated for adequate homogenization. After 8 h of enrichment (10 h for frozen FTB), a subsample of each enrichment was removed and analyzed using the Assurance® GDS for E. coli O157:H7 Tq method. Analysis was performed by both the manual and automated DNA extraction (PPMX) procedures for beef trim and finely textured beef. Analysis was also performed in both formats of Amplification tubes (standard and HT), except for green onions. Regardless of presumptive results, all test portions were confirmed following the appropriate reference method at their prescribed (reference method) enrichment times. For the USDA-FISIS MLG 5C.00 reference method, thirty 25 g test portions were enriched with 225 ± 4.5 mL of modified Tryptic Soy Broth (mTSB). Samples were macerated for adequate homogenization and incubated for 15–24 h at 42 ± 1°C. Individual carcass cloths were enriched with 200 mL of mTSB. After incubation, all samples were screened for the stx 2/1 and eae genes using the probes and primers indicated in USDA-FSIS MLG 5C.00. Samples that tested PCR-negative for the stx and/or eae gene were reported as negative. All samples that tested positive for stx and eae genes were culturally confirmed by IMS isolation with anti- E. coli O157 magnetic beads (Abraxis, Warminster, PA). Following the IMS procedure, samples were plated onto modified Rainbow® agar (mRBA) and incubated at 35 ± 2°C for 20–24 h. Upon completion of incubation, the mRBA plates were examined for colonies and latex agglutination was performed to confirm E. coli O157:H7 colonies. Samples that had no growth or were agglutination-negative on mRBA plates were reported as negative. Colonies that were agglutination-positive were streaked to SBA for isolation. SBA plates were incubated at 35 ± 2°C for 20–24 h. Following incubation, latex agglutination (Remel, San Diego) was performed for H7 on the colonies. The colonies were then analyzed by PCR for presence of stx and eae genes using the probes and primers indicated USDA-FSIS MLG 5C.00, as well as final confirmation of isolates by bioMérieux API 20E. USDA-FSIS MLG 5C.00 Method

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