PhageDx™ E. coli O157:H7 Assay in Ground Beef Level 3 Modification to Add 375 g Beef Trim and New Method Procedure for 25 g Ground Beef: AOAC Performance Tested Method(SM) 081601

BACKGROUND: ThePhageDx(™) E. coli O157:H7 Assay originally required a 5 h enrichment period and a 10 mL test sample for 25 g ground beef. The proposed method modifications seek to include beef trim (375 g) matrix and a new method procedure for 25 g ground beef comprised of 6–7 h enrichment and 1 mL test sample. OBJECTIVE: To validate the method modifications for PhageDx E. coli O157:H7 Assay to include beef trim (375 g) matrix and modify enrichment time and test sample size for 25 g ground beef. METHOD: For both matrixes, pre-warmed tryptic soya broth (TSB; 42 ± 1°C) was added in a 3:1 (media:sample size) ratio, blended, and enriched for 9–10 h (beef trim, 375 g) or 6 h (ground beef, 25 g) at 42 ± 1°C. One milliliter samples were transferred to a microfuge tube, centrifuged, and the supernatant removed. The pellet was resuspended in 0.2 mL of TSB and phage reagent was added. Samples were incubated for 2 h at 37 ± 1°C. After infection, the sample was centrifuged, and 150 µl of the supernatant was transferred to a 96-well plate. Then, lysis buffer and luciferase substrate were added and the sample read on a luminometer to determine the presence or absence of E. coli O157:H7. RESULTS: No significant differences were found between the PhageDx E. coli O157:H7 Assay method modifications and the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) reference method. CONCLUSIONS: The independent study demonstrated that the PhageDx E. coli O157:H7 Assay method modifications meet the qualifications for Performance Tested Method (SM) status. HIGHLIGHTS: The PhageDx E. coli O157:H7 Assay is a rapid detection method capable of detecting E. coli O157:H7 in 25 g ground beef and 375 g beef trim samples in as little as 8.5 h and 11.5 h total turn around time, respectively.