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Background The Simultaneous Multiplex Real Time PCR (SIMUL-qPCR) Listeria species and monocytogenes Assay is a quick, reliable method for detecting Listeria species and monocytogenes in environmental and food samples. The assay multiplexes several targets in one run to properly identify Listeria species and monocytogenes. The assay uses proprietary medium, Listeria Recovery and Enrichment Broth (LREB), for enrichment purposes. LREB was specifically formulated to improve the recovery and growth of Listeria while inhibiting competing background flora. Objective This report details the method validation study to validate frankfurters, ready-to-eat (RTE) sliced turkey, soft fresh raw cheese, chicken salad, ice cream, cooked eggs, pasteurized milk, and frozen/cooked shrimp, as well as environmental surface sponges and swabs for stainless steel, plastic, rubber, ceramic tile, and sealed concrete. Method Matrix studies, inclusivity/exclusivity, product consistency/stability, and robustness testing were conducted to assess the method’s performance. Results There were no statistically significant differences found between the candidate and reference methods in the matrix studies. Inclusivity/exclusivity testing showed that the assay was able to detect both Listeria species and monocytogenes strains while excluding the non-Listeria isolates. Small variations in critical test parameters (enrichment time, extraction reagent volume, and extracted sample volume) did not adversely affect the assay’s performance, and stability testing indicated consistent results for at least 1 year. Conclusions The data presented in this report show that this a reliable method for detecting Listeria species and monocytogenes. Highlights This assay allows for one sample to be tested for both Listeria species and monocytogenes with one PCR test.

Background The Molecular Environmental Monitoring Program (MEMP) Listeria Assay is a quick, reliable method for detecting Listeria species in environmental samples. The assay incorporates a real-time PCR approach to identifying Listeria cells expressed from the swab sample. The assay does not require an enrichment step, leading to much faster time to a negative result. Objective This report details the method validation study to validate the MEMP using environmental surface swabs for stainless steel, plastic, rubber, ceramic tile, and sealed concrete. Method Matrix studies, inclusivity/exclusivity, product consistency and stability, and robustness testing were conducted to assess the method’s performance. In the matrix studies, this method was compared to the US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 for environmental surface sponges and swabs. Results Inclusivity and exclusivity testing showed that the MEMP Listeria Assay was able to detect all 75 Listeria strains tested while excluding the 30 non-Listeria species. There were no statistically significant differences found between the candidate and reference methods. Small variations in critical test parameters (volume of extraction reagent and volume of extracted DNA sample) did not adversely affect the assay’s performance, and stability testing indicated consistent results for at least 1 year. Conclusions The data presented in this report show that the candidate method performed as well as the reference method and can therefore be used in place of the reference method for detecting Listeria species. Highlights The MEMP Listeria Assay requires no enrichment for detecting Listeria on environmental surfaces.

Background The Molecular Environmental Monitoring Program (MEMP) Salmonella Assay is a quick and reliable method for detecting Salmonella species in environmental samples. The assay incorporates a real-time PCR approach to identifying Salmonella cells expressed from the swab sample. The assay does not require an enrichment step, leading to much faster time to a negative result. Objective This report details the method validation study to validate the MEMP using environmental surface swabs for stainless steel, plastic, rubber, ceramic tile, and sealed concrete. Method Matrix studies, inclusivity/exclusivity, product consistency and stability, and robustness testing were conducted to assess the method’s performance. In the matrix studies, this method was compared to the US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 for environmental surface sponges and swabs. Results Inclusivity/exclusivity testing showed that this assay was able to detect all 100 Salmonella strains tested while excluding the 30 non-Salmonella species. There were no statistically significant differences found between the candidate and reference methods. Small variations in critical test parameters (volume of extraction reagent and volume of extracted DNA sample) did not adversely affect the assay’s performance, and stability testing indicated consistent results for at least one year. Conclusions The data presented in this report show that the candidate method performed as well as the reference method; therefore, it can be used in place of the reference method for detecting Salmonella species. Highlights The MEMP Salmonella Assay is the first and only AOAC Performance Tested MethodSM approved for detecting Salmonella on surfaces without enrichment.

Background The Simultaneous Multiplex (SIMUL) Real-Time PCR (qPCR) Salmonella Assay is a quick, reliable method for detecting Salmonella species in environmental and food samples. The assay multiplexes several targets in one run to identify Salmonella species. For most matrixes, the assay uses buffered peptone water for enrichment purposes. However, the assay can be used in conjunction with the SIMUL-qPCR Top 7 STEC Assay for raw beef trim and raw ground beef when proprietary media (Enterohemorrhagic E. coli Recovery and Enrichment Broth or EREB) has been used. This media is optimized for single-step enrichment and recovery of enterohemorrhagic E. coli and Salmonella in those matrixes. Objective This report details the method validation study to validate raw beef trim, raw ground beef, fresh raw ground poultry, ready-to-eat cooked poultry, dry pet food, pasteurized liquid eggs, peanut butter, frankfurter/sausage, poultry carcass rinse, and environmental surface sponges and swabs for stainless steel, plastic, rubber, ceramic tile, and sealed concrete. Method Matrix studies, inclusivity/exclusivity, product consistency/stability, and robustness testing were conducted to assess the method’s performance. Results Inclusivity/exclusivity testing showed the SIMUL-qPCR Salmonella Assay was able to detect Salmonella strains while excluding the non-Salmonella isolates. There were no statistically significant differences found between the candidate and reference methods in the matrix studies. Small variations in critical test parameters (enrichment time, extraction reagent volume, and extracted sample volume) didn’t adversely affect the assay’s performance, and stability testing indicated consistent results for at least one year. Conclusions The data presented demonstrate the SIMUL-qPCR Salmonella Assay is a reliable method for detecting Salmonella. Highlights The SIMUL-qPCR Salmonella Assay is validated to detect Salmonella in a variety of matrices in as little as 16 hours in Buffered Peptone Water. The assay can also be used in conjunction with the SIMUL Top 7 Assay to test for both Salmonella and the Top 7 Shiga toxin producing E. coli using a single enrichment in EREB media.

Background The Simultaneous Multiplex Real Time PCR (SIMUL-qPCR) Top 7 STEC Assay Collection is a quick, reliable method for detecting top seven Shiga toxin-producing Escherichia coli (STEC) in raw beef trim, raw ground beef, and beef carcass sampling sheets. Each assay multiplexes several targets in one run to identify E. coli O157: H7, O26, O45, O103, O111, O121, O145, Shiga toxin, and intimin genes. This collection uses specifically optimized proprietary media for single-step recovery and enrichment of enterohemorrhagic E. coli. Objective This report details the method validation study to validate raw beef trim, raw ground beef, and beef carcass sampling sheets. Method Matrix studies for raw beef trim, raw ground beef, and beef carcass sampling sheets, inclusivity/exclusivity, product consistency/stability, and robustness testing were conducted to assess the method’s performance. Results Fifty top seven STEC isolates were analyzed with the SIMUL-qPCR assay. Thirty-two isolates, including closely related non-E. coli species and E. coli non-STEC strains, were also tested. Inclusivity showed the collection detected the Shiga toxin (stx) gene, intimin (eae) gene, and top seven serogroups. None of the 32 exclusivity strains were detected. The candidate and reference methods’ results had no statistically significant differences during matrix studies. Small variations in critical test parameters (enrichment time, extraction reagent volume, and extracted sample volume) did not adversely affect the assay’s performance, and stability testing indicated consistent results for at least one year. Conclusions The data presented demonstrate that the SIMUL-qPCR Top 7 STEC Assay is a reliable method for detecting the top seven STEC. Highlights The Applied Food Diagnostics, Inc. Simultaneous Multiplex Real Time PCR (SIMUL-qPCR) Top 7 STEC Assay is capable of detecting the top seven Shiga toxin-producing Escherichia coli in beef trim, ground beef, and beef carcass sampling sheets in as little as 10 hours of enrichment.