Immunomagnetic separation and visual fluorescence detection of E. coli O157 using AOAC approved SAS Molecular Tests.

Abstract

The SAS Molecular Tests method for the detection of E. coli O157 in various food matrixes has been certified by the AOAC Research Institute and designated Performance Tested Method No. 031203. The current method modification includes the optional immunomagnetic separation (IMS) to enrich the bacteria as well as optional visual fluorescence readout without the use of a turbidimeter. The following study was conducted to validate the proposed modifications against the U.S. Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook (MLG) and the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) reference methods. E. coli O157:H7 ATCC 35150 and NM (non-motile) 700377 strains were used to inoculate the ground beef, beef trim, and spinach to obtain 20 low (0.23-2 CFU/test portion) and five high levels (3-5 CFU/test portion) of inoculations. Enriched samples were tested directly and subjected to anti-E. coli IMS prior to the SAS Molecular Tests. Results were determined via visual fluorescence and via turbidity using a turbidimeter. All the replicates, irrespective of the results, were confirmed using MLG 5.05 or BAM Chapter 4A methods. Results indicated that there were no significant differences in the detection of fractional positives (5-15 positives out of 20 replicates) with any of the methods tested above as compared to the reference methods. No false positives or negatives were detected except for two in the ground beef with IMS+Turbidity method. No false-negative samples were detected. Statistical analysis indicated that the modified methods were equivalent to the reference methods in detecting E. coli O157:H7 in the food matrixes tested. SAS Molecular Tests E. coli O157 Detection Kit can be used to detect E. coli O157:H7 in ground beef, beef trim, and spinach. The inclusion of IMS in the modified method improved the detection rate of E. coli O157:H7 in spinach and showed comparable detection rate in ground beef and beef trim. The optional use of visual fluorescent reagent and heat block either with IMS or without IMS produced results that were comparable to the results obtained from using a real-time lurbidimeter.