ERP Micro December 2019

FDA BAM Chapter 4A Method

For the FDA BAM 4A reference method, thirty 25 g test portions were enriched with 225 mL of modified Buffered Peptone Water with pyruvate and Acriflavin-Cefsulodin-Vancomycin (ACV) supplement (mBPWp). Twenty-five g of food was weighed into 225 mL of mBPWp, then macerated for adequate homogenization. The homogenate was incubated at 37°C ± 1°C static for 5 h, then 1 ml each of the ACV supplements was added per 225 mL of mBPWp and incubated at 42°C ± 1 °C static overnight (18-24 h). After incubation, all sample enrichments were serially diluted in Butterfield’s phosphate diluent buffer (BPBD) to 10 -2 and 10 -4 dilutions. These dilution levels were spread-plated onto Tellurite Cefixime – Sorbitol MacConkey Agar (TC-SMAC) and R&F® E . coli O157:H7 Agar (RAO157). Plates were incubated at 37 ± 1° C for 18-24 h. Upon completion of incubation, the TC-SMAC and RAO157 plates were examined for typical colonies and latex agglutination was performed to confirm E. coli O157:H7 colonies. Samples that had no growth or were agglutination-negative were reported as negative. Colonies that were agglutination-positive were streaked to TSA-YE for isolation. TSA-YE plates were incubated at 37°C ± 1°C for 18 - 24 h. Following incubation, latex agglutination was performed for O157 and H7 on the colonies (Remel, San Diego, CA). The agglutination-positive colonies were then confirmed to be E . coli by bioMérieux API 20E. Colonies which agglutinated with both O157 and H7 and biochemically confirmed to be E . coli , were determined to be E. coli O157:H7. Statistical Analysis The probability of detection (POD) was calculated as the number of positive outcomes divided by the total number of trials (14). The POD was calculated for the candidate presumptive results, POD CP, the candidate confirmatory results, POD CC , the difference in the candidate presumptive and confirmatory results, dPOD CP, presumptive candidate results that confirmed positive, POD C, the reference method, POD R , and the difference in the confirmed candidate and reference methods, dPOD C . For food and carcass cloth testing, the candidate method was evaluated using both the manual and automated sample processing methods. Additionally, both amplification tube size formats were tested for each DNA extraction method. Inclusivity Evaluation - PPMX This protocol examined the ability of the GDS EHEC to detect a variety of the claimed E. coli O157:H7 and to distinguish those strains from closely related E. coli non-O157:H7 strains and non- E. coli species through sample enrichment as directed by the GDS EHEC Directions for Use (DFU) (4). Inclusivity and exclusivity were previously performed for GDS EHEC assay. For a minor modification including a new instrument (PPMX), inclusivity and exclusivity are also required to

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