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Background Frequent testing for Legionella concentration in water is required by most health risk monitoring organizations worldwide. Domestic hot water and cooling tower water networks must be regularly controlled to prevent Legionnaires’ disease, a potentially deadly lung infection. MICA Legionella is the fastest culture-based detection method for all serogroups of Legionella pneumophila, with automatic enumeration in 48 h and no need for confirmation. Objective This study compares the performance and robustness of MICA Legionella with the reference method ISO 11731:2017 for the enumeration of culturable L. pneumophila. Methods MICA Legionella and ISO 11731:2017 results were compared for domestic hot water and cooling tower water. Inclusivity and exclusivity were tested on reference and environmental strains. Ruggedness, lot-to-lot consistency, and stability of the reagents kit were also studied. Results Enumeration of L. pneumophila by MICA Legionella was statistically equivalent to ISO 11731:2017 in both matrixes. In cooling tower waters, MICA Legionella showed better sensitivity than ISO 11731:2017. It presented a 94% sensitivity and a 97% specificity. Conclusion MICA Legionella is a highly sensitive and specific method for culturable L. pneumophila enumeration. It presents, in 48 hours, equivalent or better results than ISO 11731:2017. Its protocol is robust to variations. Its reagents kit is stable for up to 18 months. Highlights MICA Legionella is a robust and reliable method for the enumeration of culturable L. pneumophila in domestic and cooling tower water. It reduces significantly the number of sample pretreatments required in ISO 11731:2017. Automatic identification and enumeration of L. pneumophila microcolonies eliminates the requirement to have skilled analysts and limits the results variability. It also greatly reduces the time to results to 48 h instead of 7–10 days with ISO 11731:2017 while providing statistically equivalent results.

Background The Thermo Scientific SureTect™ Campylobacter jejuni, C. coli, and C. lari PCR Kit is a real-time PCR assay for the detection and differentiation of C. jejuni, C. coli, and C. lari from raw poultry, ready-to-cook poultry products, and environmental samples. Objective The Thermo Scientific SureTect Campylobacter jejuni, C. coli, and C. lari PCR Kit was evaluated for AOAC®Performance Tested MethodsSM certification. Methods Inclusivity/exclusivity, matrix studies, product consistency and stability, and robustness testing were conducted to assess the method’s performance. In the matrix studies, the method was validated against United States and international reference methods for Campylobacter detection. Results There were no statistically significant differences found in the matrix studies between the candidate and reference methods when analyzed by probability of detection. All 52 inclusivity strains and none of the 51 exclusivity strains tested were detected by the assay. Robustness testing demonstrated that the assay gave reliable performance with specific method deviations outside of the recommended parameters, and the real-time stability testing demonstrated that there were no statistically significant differences between kit lots, validating the stated shelf life of the kit. Conclusion The data presented support the product claims that the Thermo Scientific SureTect Campylobacter jejuni, C. coli, and C. lari PCR assay is suitable for the detection and differentiation of C. jejuni, C. coli, and C. lari from raw poultry, ready-to-cook poultry products, and environmental samples. Highlights Presumptive results can be obtained in as little as 23 h. Microaerophilic incubators are not required for enrichment.

Background The Thermo Scientific SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay are real-time PCR kits for the rapid detection of E. coli O157:H7 and non-E. coli O157 Shiga toxin-producing E. coli (STEC) serotypes (O26, O45, O103, O111, O121, O145) from fresh raw spinach, fresh baby leaves, fresh cut tomatoes, frozen raw beef, raw beef trim, and beef carcass sponges. Objective Both assays were evaluated for AOAC®Performance Tested MethodsSM certification. Methods Detection and confirmation inclusivity/exclusivity, matrix, product consistency and stability, and robustness studies were conducted. In the matrix studies, the candidate method was validated against United States and international reference methods for STEC serotypes. Results Matrix studies showed no statistically significant differences between the candidate and reference method results when analyzed by probability of detection. For each inclusivity/exclusivity study, all inclusivity strains and no exclusivity strains were detected by either kit. Robustness testing demonstrated that the identification assay performed reliably despite method deviations; however, although not statistically significant, the screening assay performance was impacted. Product consistency and stability testing demonstrated no statistically significant differences between kit lots and storage time points. Conclusion The data presented show that both assays constitute a rapid and reliable workflow for the detection and confirmation of E. coli O157:H7 and stipulated non-E. coli O157:H7 STEC serotypes from the tested matrixes. Highlights Results are obtained in 80 min post-enrichment with both assays run simultaneously, allowing for the detection and confirmation of STEC within a single workflow.

Background The Thermo Scientific™ SureTect™ Salmonella species PCR Assay utilizes Solaris™ reagents for performing PCR for the rapid and specific detection of Salmonella species in a broad range of foods and select environmental surfaces. Objective The aims were to demonstrate the reproducibility of the Thermo Scientific SureTect Salmonella species PCR Assay in a collaborative study using a challenging matrix, cocoa powder, and to extend the scope of the method. Method In the collaborative study, the candidate method was compared to the US Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). Fourteen participants from nine laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Three levels of contamination were evaluated for each matrix. Statistical analysis was conducted according to the probability of detection statistical model. In addition, 11 matrix studies were performed comparing the candidate method to the FDA/BAM Chapter 5 or US Department of Agriculture, Food Safety and Inspection Service, Microbiology Laboratory Guidebook 4.10 Isolation and Identification of Salmonella from Meat, Poultry, Pasteurized Egg, and Siluriformes (Fish) Products and Carcass and Environmental Sponges reference method. Nine of these matrices were also compared to the EN ISO 6579–1:2017/Amd.1:2020(E) Microbiology of the food chain—Horizontal method for the detection, enumeration and serotyping of Salmonella—Part 1: Detection of Salmonella spp.—AMENDMENT 1: Broader range of incubation temperatures, amendment to the status of Annex D, and correction of the composition of MSRV and SC reference method. Results In the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated, and the method demonstrated acceptable interlaboratory reproducibility as determined in the collaborative evaluation. False-positive and false-negative rates were determined for the matrix and produced values of Conclusions Based on the data generated, the method demonstrated acceptable interlaboratory reproducibility data and statistical analysis. Highlights Due to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 375 g cocoa powder is known to be a challenging matrix for PCR methods. No unusual cross-contamination or false-positive/negative was reported, highlighting the ease of use, reproducibility, and robustness of the method.

Background The AquaCHROM™ ECC method from CHROMagar is intended for the detection and enumeration of Escherichia coli and coliform bacteria in 100 mL water samples after 18–24 h of incubation at 35–37°C. Objective To validate the AquaCHROM ECC method for qualitative and quantitative detection of E. coli and coliforms with different water matrixes. Methods Inclusivity/exclusivity studies were conducted. AquaCHROM ECC was compared to U.S. cultural reference methods in unpaired matrix studies for detection of E. coli and coliforms in tap water, well water, lake water, and bottled water, and for enumeration in tap water, well water, and lake water. Three production lots of AquaCHROM ECC were tested for product consistency and stability. Variations in incubation time and temperature were evaluated in robustness testing. Results Inclusivity/exclusivity results demonstrated expected performance with the exception of three strains of Salmonella enterica, two species of Shigella, and one strain of Aeromonas, which turned the media blue or yellow. Results from the matrix studies demonstrated that AquaCHROM ECC and the reference methods are not statistically different for detection of E. coli and coliforms and statistically equivalent for enumeration of E. coli and coliforms. The AquaCHROM ECC powder production was proven to be consistent with a 24-month shelf life. Variation in temperature did not affect the method performance. Shortening the incubation time is not recommended. Conclusion AquaCHROM ECC is an effective method for the detection and enumeration of E. coli and coliforms for the water matrixes evaluated. Highlights The AquaCHROM ECC method is a quick, one-step method for the recovery and enumeration of E. coli and coliforms in 100 mL water samples. It is a non-agar-based chromogenic medium which provides a clear result without the use of a UV lamp.

Background The Assurance® GDS for Cronobacter Tq II assay is a nucleic acid amplification system for the qualitative detection of Cronobacter. The method uses an upfront concentration of the target organism from the enrichment by immunomagnetic separation (IMS) using the PickPen® device. Objective The Assurance GDS for Cronobacter Tq II method was evaluated for Official Methods of AnalysisSM certification. Method The matrix was compared to the ISO 22964:2017: Microbiology of the Food Chain—Horizontal Method for the Detection of Cronobacter spp. standard and using an alternative confirmation procedure. The alternative method was evaluated using 10 g test portions in an unpaired study design for powdered infant formula (milk based with iron and DHA) containing probiotics. Eleven technicians from eight laboratories located within the United States and Europe participated in the collaborative study. Statistical analysis was conducted according to the probability of detection (POD) statistical model as presented in the AOAC validation guidelines. The difference in laboratory POD (dLPODC) values with 95% confidence intervals across collaborators was calculated for each level between the candidate and reference method results and between the candidate presumptive and confirmed results. Results Results obtained for the low inoculum level test portions produced a dLPOD value with a 95% confidence interval of 0.03 (−0.18, 0.15). The dLPOD results indicate equivalence between the candidate method and reference method for the matrix evaluated. The method also demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. There were no false negative results; the false positive rate was determined and produced a value of Conclusions Based on the data generated, the method demonstrated Assurance GDS for Cronobacter Tq II assay produced acceptable interlaboratory reproducibility data and statistical analysis. Highlights The Assurance GDS for Cronobacter Tq II method is suitable for the rapid qualitative detection of Cronobacter in infant formulas, infant cereals, ingredients, and environmental samples.

Background The PathogenDx family of assays uses microarray technology to simultaneously detect the presence of bacterial and fungal pathogens in food products, environmental surfaces, and cannabis products. Objective The Detectx Combined assay was validated for the detection of Aspergillus, (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus), Salmonella, and a broad range of STEC (stx1 and/or 2) species. The validation consisted of two matrix studies in dried hemp flower and dried cannabis flower (>0.3% delta-9 tetrahydrocannabinol) flower, product consistency, stability, robustness, and inclusivity and exclusivity for two targets: Aspergillus and STEC. Method The PathogenDx Detectx Combined assay was evaluated with 30 replicates in each matrix and confirmed according to the instructions outlined in this study. Results Results of the validation study met the requirements of AOAC Standard Method Performance Requirement (SMPR®) 2020.002 and 2020.012. In the inclusivity and exclusivity study, all target isolates (Aspergillus and STEC) were correctly detected. For the exclusivity study, 26 out of 30 Aspergillus and 30 out of 30 STEC non-target strains were correctly excluded. In the matrix study, the PathogenDx Detectx Combined assay showed no significant statistical differences between confirmed results for dried hemp and cannabis flower. Robustness testing indicated that small changes to the method parameters did not impact the performance of the assay. Stability and consistency studies verified that the assay’s shelf-life claims were appropriate, and manufacturing of the assay was consistent. Conclusions The validation study indicated that the PathogenDx DetectX Combined assay was successful in detection of the new target analytes (Aspergillus flavus, Aspergillus fumigatus, Aspergillus niger, and Aspergillus terreus and STEC containing stx1 and/or 2) and could successfully recover these organisms and Salmonella from dried hemp flower and dried cannabis flower (>0.3% delta-9 tetrahydrocannabinol). Highlights The PathogenDx DetectX Combined Assay will be the first PTM approved multiplex assay for Aspergillus, E. coli and Salmonella that does not require an enrichment step.

The Thermo Scientific™ SureTect™ Listeria monocytogenes PCR Assay uses Solaris reagents for performing PCR for the rapid and specific detection of Listeria monocytogenes in a broad range of foods and selected environmental surfaces.To demonstrate reproducibility of the SureTect Listeria monocytogenes PCR Assay in a collaborative study using a challenging matrix, full-fat cottage cheese (25 g). To extend the scope of the method.In the collaborative study, the candidate method was compared to the United States Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 10 Listeria reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). Eighteen participants from 10 laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Three levels of contamination were evaluated for each matrix. Statistical analysis was conducted according to the probability of detection (POD) statistical model. In addition, to extend the scope, six matrix studies were performed comparing the candidate method to the FDA/BAM reference method. One of these matrixes was also compared to the ISO 11290-1:2017 Microbiology of the Food Chain-Horizontal Method for the Detection and Enumeration of Listeria monocytogenes and of Listeria spp.-Part 1: Detection Method Reference Method.In the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated, and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. The two PCR instruments used in the study performed equivalently. All presumptive positives were confirmed via the alternative confirmation procedure. In the pre-collaborative studies, the results showed comparable performances between the candidate method and the reference method for all matrixes tested.Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis.Due to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 25 g full-fat cottage cheese is known to be a challenging matrix to test. No unusual cross-contamination or false positive/negative data were reported, highlighting the ease of use, reproducibility, and robustness of the method.

The Thermo Scientific SureTect™ Listeria species PCR assay utilizes SolarisTM reagents for performing PCR for the rapid and specific detection of Listeria species in a broad range of foods and selected environmental surfaces.To demonstrate reproducibility of the Thermo Scientific SureTect Listeria species PCR assay in a collaborative study using a challenging matrix, full-fat cottage cheese (25 g), to extend the scope of the method.In the collaborative study, the candidate method was compared to the US Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Ch. 10 Listeria reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). The candidate method included its own confirmation procedure. Eighteen participants from 10 laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Statistical analysis was conducted according to the probability of detection (POD) statistical model. In addition, in order to extend the scope of the method, seven matrix studies were performed comparing the candidate method to the FDA/BAM reference method. One of these matrixes was also compared to the ISO 11290-1:2017 Microbiology of the food chain-Horizontal method for the detection and enumeration of Listeria monocytogenes and of Listeria spp.-Part 1: Detection method reference method.In the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. The two PCR instruments used in the study performed equivalently. All presumptive positives were confirmed via the alternative confirmation procedure. In the pre-collaborative studies, the results showed comparable performances between the candidate method and the reference method for all matrixes tested.Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis.Due to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 25 g full-fat cottage cheese is known to be a challenging matrix to test. No unusual cross-contamination, or false-positive/negative data was reported, highlighting the ease of use, reproducibility, and robustness of the candidate method.

Background Reveal® 3-D for Peanut is an immunochromatographic, lateral flow test for qualitative detection of peanut residue in food manufacturing and food preparation settings. The test can detect low ppm levels of peanut in clean-in-place (CIP) rinses and in swabs from environmental surfaces and can serve as a tool in managing allergen risk. Objective The objective of the study was to validate the lateral flow method for detection of peanut in CIP rinses, specifically water, peroxyacetic acid/hydrogen peroxide, and quaternary ammonium compound rinses, and in swabs taken from stainless steel and plastic surfaces. Methods CIP rinses spiked with low levels of peanut were tested, as were surfaces inoculated with peanut. Specificity and assay interference were assessed in testing of food commodities with and without added peanut. Assay robustness and test kit stability and consistency testing were also performed. Results Results demonstrated that the lateral flow test can detect peanut in CIP rinses in the range of 2–4 ppm and in environmental surface swabs in the range of 3–4 µg/100 cm2. Results of specificity testing with 29 common food items showed lack of cross-reactivity, and potential assay interference only from walnut. Data from stability trials supports expiration dating for the kit of up to 23 months post-manufacture. Conclusions and Highlights The lateral flow test is a sensitive, specific, and rapid method for detection of low levels of peanut residue in CIP rinses and environmental samples and can be an important component in a comprehensive allergen risk management program.

Testing milk for antibiotics before acceptance into dairies is required by the U.S. Pasteurized Milk Ordinance. Technological advances in tests have reduced screening times and improved detection accuracy. This work describes the validation of the Charm Rapid One Step Assay Beta-Lactam 30 Second Test according to the U.S. Food and Drug Administration Center for Veterinary Medicine protocol for raw commingled milk. Milk is added to the lateral flow test strip in an incubator/reader to deliver a 30 second result. Independent laboratory validation followed sensitivity, interference, and incurred residue protocols. Sensitivity, in parts per billion (ppb = µg/kg), using a probit curve determined 90% percent detection with 95% confidence, which met National Conference of Interstate Milk Shipments (NCIMS) specifications. Six U.S. approved beta-lactam drugs were detected below, but within 50% of, target/tolerance levels for penicillin G 2.9 ppb, ampicillin 5.9 ppb, amoxicillin 5.8 ppb, cephapirin 13 ppb, cloxacillin 8.1 ppb, and ceftiofur metabolites 73 ppb. No interferences were observed from 33 animal drugs at 100 ppb, somatic cells at 1.2 million/mL, or bacterial levels of >300 000 CFU/mL. Incurred residue detection levels were similar to levels determined with the spiked parent compound. The data support NCIMS that the BL30SEC method met U.S. criteria for testing milk for beta-lactams.

Background: NeoSeekTM STEC is a single-source, service-based method for detection and identification of select Shiga toxin–producing Escherichia coli (STEC), including E. coli O157:H7 and STEC of somatic groups O26, O45, O103, O111, O121, and O145. The method is a multiplex molecular method utilizing more than 80 genetic targets to identify STEC in complex matrices such as food enrichment cultures. Objective: A study was conducted to validate the NeoSeek method for detection of select STEC in raw beef trim. Methods: Performance of the NeoSeek STEC method was compared with that of the U.S. Department of Agriculture, Food Safety and Inspection Service reference methods for E. coli O157:H7 and non-O157 STEC for detection of E. coli O157:H7 and E. coli O26:H11 in raw beef trim. Additionally, inclusivity/exclusivity testing and method robustness testing were performed. Results: Results of raw beef trim testing showed no statistically significant differences in performance between the NeoSeek and reference methods in the ability to detect either E. coli O157:H7 or E.coli O26:H11, as determined by probability of detection analysis. Results of inclusivity and exclusivity testing showed 100% expected results with target and nontarget bacteria, with the exception of a single strain of E. coli O157:H7, which was subsequently verified to be stx-negative by PCR. Conclusions and Highlights: NeoSeek STEC is an accurate, reliable method for rapid detection and identification of select STEC in complex populations such as beef trim enrichment cultures.

Background: The Bio-Rad iQ-Check Listeria spp. Kit uses real-time PCR technology for detection of Listeria species in select food matrixes and environmental surfaces. Objective: The iQ-Check Listeria spp. method was modified to reduce the enrichment medium volume for environmental sponges from 225 and 100 to 60 mL and to reduce the enrichment time for sponges and swabs from 25 ± 1 to as short as 18 h. The modified method was validated with stainless steel, polystyrene plastic, and sealed concrete using sponges or swabs with two different neutralizing buffers (Letheen Broth and HiCap™ Neutralizing Broth). In addition, the Bio-Rad Free DNA Removal Solution was used for all environmental samples. Methods: The iQ-Check Listeria spp. modified method was compared with the reference culture method in the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapter 8.10 using an unpaired study design. Results: In the method comparison study, the iQ-Check Listeria spp. modified method demonstrated no statistical difference in performance between candidate and reference method results or between presumptive and confirmed results for all environmental surfaces analyzed using HiCap Neutralizing Broth (World Bioproducts LLC) and Letheen broth. Conclusions: The modified iQ-Check Listeria spp. method is an effective method for the detection of Listeria species in environmental surfaces using both types of neutralizing buffer. Highlights: The method modification was granted based on the data collected.

The Thermo Scientific SureTect™ Campylobacter jejuni, C. coli, and C. lari PCR Kit is a real-time PCR assay for the detection and differentiation of C. jejuni, C. coli, and C. lari from raw poultry, ready-to-cook poultry products, and environmental samples.The Thermo Scientific SureTect Campylobacter jejuni, C. coli, and C. lari PCR Kit was evaluated for AOAC®Performance Tested MethodsSM certification.Inclusivity/exclusivity, matrix studies, product consistency and stability, and robustness testing were conducted to assess the method's performance. In the matrix studies, the method was validated against United States and international reference methods for Campylobacter detection.There were no statistically significant differences found in the matrix studies between the candidate and reference methods when analyzed by probability of detection. All 52 inclusivity strains and none of the 51 exclusivity strains tested were detected by the assay. Robustness testing demonstrated that the assay gave reliable performance with specific method deviations outside of the recommended parameters, and the real-time stability testing demonstrated that there were no statistically significant differences between kit lots, validating the stated shelf life of the kit.The data presented support the product claims that the Thermo Scientific SureTect Campylobacter jejuni, C. coli, and C. lari PCR assay is suitable for the detection and differentiation of C. jejuni, C. coli, and C. lari from raw poultry, ready-to-cook poultry products, and environmental samples.Presumptive results can be obtained in as little as 23 h. Microaerophilic incubators are not required for enrichment.

Background The Thermo Scientific™ SureTect™Staphylococcus aureus PCR Assay is a real-time PCR assay for the detection of Staphylococcus aureus in dairy samples. Objective The Thermo Scientific SureTect Staphylococcus aureus PCR Assay was evaluated for AOAC Performance Tested MethodSM certification. Methods Inclusivity/exclusivity, matrix studies, product consistency and stability, and robustness testing were conducted to assess the method’s performance. For the matrix study, the method was validated on the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument and the Applied Biosystems 7500 Fast Real-Time PCR instrument against the ISO 6888-3:2003 Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of coagulase-positive staphylococci (Staphylococcus aureus and other species)—Part 3: Detection and MPN technique for low numbers, and the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Ch. 12, Staphylococcus aureus, 2016, reference methods. Results Matrix studies showed no statistically significant differences between the candidate and reference methods or between presumptive and confirmed results. The inclusivity/exclusivity study correctly identified/excluded all strains analyzed. Robustness testing showed no statistically significant difference in assay performance after set method parameter deviations, and product consistency and stability studies demonstrated no statistically significant differences in performance between kit lots at different expiration points. Conclusion The data presented show that the assay is a rapid and reliable workflow for the detection of S. aureus from dairy matrixes. Highlights The PCR assay allows for fast, reliable detection of S. aureus in dairy matrixes with results obtained in as little as 80 min post enrichment.

The Thermo Scientific SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect Escherichia coli STEC Identification PCR Assay are real-time PCR kits for the rapid detection of E. coli O157:H7 and non-E. coli O157 Shiga toxin-producing E. coli (STEC) serotypes (O26, O45, O103, O111, O121, O145) from fresh raw spinach, fresh baby leaves, fresh cut tomatoes, frozen raw beef, raw beef trim, and beef carcass sponges.Both assays were evaluated for AOAC®Performance Tested MethodsSM certification.Detection and confirmation inclusivity/exclusivity, matrix, product consistency and stability, and robustness studies were conducted. In the matrix studies, the candidate method was validated against United States and international reference methods for STEC serotypes.Matrix studies showed no statistically significant differences between the candidate and reference method results when analyzed by probability of detection. For each inclusivity/exclusivity study, all inclusivity strains and no exclusivity strains were detected by either kit. Robustness testing demonstrated that the identification assay performed reliably despite method deviations; however, although not statistically significant, the screening assay performance was impacted. Product consistency and stability testing demonstrated no statistically significant differences between kit lots and storage time points.The data presented show that both assays constitute a rapid and reliable workflow for the detection and confirmation of E. coli O157:H7 and stipulated non-E. coli O157:H7 STEC serotypes from the tested matrixes.Results are obtained in 80 min post-enrichment with both assays run simultaneously, allowing for the detection and confirmation of STEC within a single workflow.

Background The Clear Safety Listeria method utilizes polymerase chain reaction (PCR) amplification and targeted next-generation sequencing technology to detect Listeria species (L. monocytogenes, L. innocua, L. ivanovii, L. marthii, L. grayi, L. welshimeri, and L. seeligeri) in hot dogs and on selected environmental surfaces. Objective The aim was to validate the candidate method according to current AOAC guidelines. Method The candidate method was compared to the reference method for hot dogs and the environmental surfaces. The method was also evaluated for inclusivity and exclusivity using 50 inclusivity strains and 30 exclusivity strains for each reported target. Product consistency and stability was tested and robustness was evaluated with changes in enrichment temperature, volume of sample treatment, and aliquot volume for PCR. Results The candidate method demonstrated no statistically significant differences using the probability of detection model between candidate and reference methods or between presumptive and confirmed results for all environmental surfaces and hot dogs. Additionally, the candidate method detected all inclusivity organisms and excluded all exclusivity organisms for each reported target. Product lots were shown to be consistent and data supported the kit’s shelf life. Finally, the robustness study demonstrated no statistical differences when the volume of sample or the aliquot volume for PCR was altered. Increasing the incubation temperature to 37 ± 1 °C resulted in greater recovery of L. monocytogenes as compared to 35 ± 1 °C and 30 ± 1 °C. Conclusions The Clear Safety Listeria method is statistically equivalent to the reference methods for the detection of L. monocytogenes and Listeria spp. in hot dogs and on selected environmental surfaces. Highlights The Clear Safety Listeria method is an automated, highthroughput NGS-based method capable of detecting Listeria species in the hot dog and environmental samples within 28h.

Background: Listeria Right Now™ is a novel, enrichment-free test for the detection of Listeria spp. in swab samples taken from environmental surfaces. Results are available in less than 1 h. In a previous Performance Tested MethodSM (PTM) study, the test was validated for swab samples from stainless-steel and sealed concrete surfaces. Objective: A PTM matrix extension study was conducted to validate the method for the detection of Listeria spp. in swab samples from ceramic tile, plastic, and rubber surfaces. Methods: Performance of the Listeria Right Now method was compared to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedure for the detection of Listeria spp. in swab samples taken from inoculated ceramic tile, plastic, and rubber surfaces. Data were analyzed using a probability of detection model. Results: There were no significant differences in performance between the Listeria Right Now and reference culture methods for any of the three surfaces tested, as determined by probability of detection analysis. Conclusions: The Listeria Right Now method is an effective procedure for the detection of Listeria spp. from a variety of environmental surfaces. Highlights: Listeria Right Now provides accurate results, without enrichment, in real time. This enables food industry personnel to react swiftly to suspected Listeria contamination incidents.

Background: The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight MS for the rapid and accurate identification and confirmation of Gram-negative bacteria from select media types. The alternative method was evaluated in a method extension study of AOAC INTERNATIONAL First Action Official MethodSM 2017.09 using nonselective and selective agars to identify Cronobacter spp., Salmonella spp., Campylobacter spp., and select Gram-negative bacteria. Results obtained by the Bruker MALDI Biotyper were compared to the traditional biochemical methods as prescribed in the appropriate reference methods. Methods: Two collaborative studies were organized, one in the United States focusing on Cronobacter spp. and other Gram-negative bacteria and one in Europe focusing on Salmonella spp. and other Gram-negative bacteria. Fourteen collaborators from seven laboratories located within the United States participated in the first collaborative study for Cronobacter spp. Fifteen collaborators from 15 service laboratories located within Europe participated in the second collaborative study for Salmonella spp. For each target organism (either Salmonella spp. or Cronobacter spp.), a total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Cronobacter spp. or Salmonella spp.) and 8 exclusivity organisms (non-Cronobacter spp. and non-Salmonella spp. closely related Gram-negative organisms). For the Campylobacter spp. method extension, 17 collaborators from eight laboratories located within the United States (seven laboratories) and Canada (one laboratory) participated in the collaborative study. A total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Campylobacter spp.) and 8 exclusivity organisms (non-Campylobacter spp. closely related Gram-negative organisms). Results: After testing was completed, the total percentage of correct identifications from each agar type for each strain was determined at a percentage of 100.0% to the genus level for the Cronobacter study and a percentage of 100.0% to the genus level for the Salmonella study. For the Campylobacter method extension, a correct identification and confirmation rate of 100.0% was obtained for the Campylobacter organisms at the species level. For non-Cronobacter, non-Salmonella, and non-Campylobacter organisms, 100.0% were correctly identified. Conclusions: The results indicated that the alternative method produced equivalent results when compared to the confirmatory procedures specified by each reference method. Highlights: The method extension can be modified to include the identification and confirmation of Campylobacter jejuni, Campylobacter coli, and Campylobacter lari.

Background: The MC-Media Pad™ Yeast and Mold (YM) is a ready-to use culture device that combines a test pad coated with medium and water-absorption polymers that is designed for the rapid quantification of yeast and mold in food products. Objective: The MC-Media Pad YM was compared with the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) for the enumeration of yeast and mold in frozen orange juice concentrate. Methods: The candidate method was evaluated using a paired study design in a multilaboratory collaborative study following the current AOAC Validation Guidelines. Three target contamination levels (low, 10–100 CFU/g; medium, 100–1000 CFU/g; and high 1000–10 000 CFU/g) and an uninoculated control level (0 CFU/g) were evaluated. MC-Media Pad YM devices were enumerated after 48 and 72 h of incubation. Results: Plate count obtained by both methods were log-transformed and the difference of means (including 95% confidence intervals), repeatability SD, and SD were determined for each contamination level. Conclusions: No statistical difference was observed between the MC-Media Pad YM (for both 48 and 72 h) and the FDA BAM for each contamination level. Highlights: The new method offers a convenient alternative to the reference method (FDA BAM) for detection of yeast and mold contamination in food products, yielding reliable and comparable results in 48 h compared to 5 days for the reference method.

Background: Solus One Salmonella is designed to accurately detect Salmonella species (Salmonella enterica subspecies enterica, salamae, arizonae, diarizonae, houtenae, indica, and Salmonella bongori) from select food matrixes and stainless-steel and plastic environmental surfaces. Solus One Salmonella uses an antibody-based technology test system that is paired with media and our proprietary media supplement, the Solus One Salmonella supplement combined with a manual or automated sample preparation method. Objective: Solus One Salmonella was evaluated for inclusivity and exclusivity, and a matrix comparison study was done for six food matrixes (raw beef trim, pasteurized liquid egg, raw salmon, cheddar cheese, Romaine lettuce, nonfat dry milk) and two environmental surfaces (stainless steel and polystyrene). Methods: Solus One Salmonella was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5: Salmonella (July 2018) and the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Manual, 4.09 (January 2017) in the matrix study. Both the manual and automated sample preparation methods were performed for cheddar cheese and stainless-steel environmental surfaces. Results: For the inclusivity and exclusivity evaluation, Solus One Salmonella correctly detected all 108 target organism isolates and correctly excluded all 35 nontarget strains that were analyzed. Conclusions: In the method comparison study, both Solus One Salmonella manual and automated sample preparation methods demonstrated no significant differences based on probability of detection (POD) statistical analysis between presumptive and confirmed results or between candidate and reference method results for the six food matrixes after 20–22 h and two environmental surfaces after 16–20 h of enrichment time. POD analysis of Solus One Salmonella method robustness, product consistency, and stability studies using the automated sample preparation method demonstrated no statistically significant differences.

Background: Probe4Cronobacter test kit is based on the use of a fluorescence-labeled peptide nucleic acid probe (PNA) allied to fluorescence microscopy. A sample is taken after a 24 h enrichment of rehydrated 30 g portions of powdered infant formula (PIF). The method uses ready to use dropper solutions applied directly in the sample. This simple process takes less than 2 h to provide a result. In the presence of Cronobacter species, bright red rod-shaped cells will be visible under a fluorescence microscope. Objective: Probe4Cronobacter validation as a new method for the detection Cronobacter species in Powdered Infant Formula (PIF) under the AOAC Performance Tested MethodsSM (License No. 081702). Methods: The validation study encompassed matrix comparison study, inclusivity and exclusivity testing and robustness studies (stability, kit variation, and ruggedness). Results: The inclusivity and exclusivity testing (50 and 35 strains, respectively) yielded no false negative or false positive results. Probe4Cronobacter was compared to the ISO/TS 22964:2006 in 30 g of PIF samples within method comparison in an unpaired study. A total of 30 samples with both low and high level of inoculation were analyzed by Probe4Cronobacter and compared to the same number of samples screened by ISO/TS 22964:2006. No statistically significant differences between presumptive and confirmed results or between candidate and reference method results were observed. Robustness studies showed a high level of consistency and integrity of the kit when different parameters were varied. The deviation conditions tested did not affect the performance of the kit. Conclusions: Probe4Cronobacter test kit has shown to be a accurate, highly sensitive and robust methods for the detection of Cronobacter spp. in PIF samples.

Background: Listeria Right NowTM is a novel, enrichment-free molecular method for detection of Listeria spp. in swab samples from environmental surfaces. The test provides results in real time, indicating the current or recent presence of Listeria spp. in the environment. After sampling, the entire contents of the swab are subject to sample processing, releasing large quantities of target ribosomal RNA molecules into the lysate. A portion of the lysate is then tested using the ANSR® for Listeria isothermal nucleic acid amplification assay. Objective: A Performance Tested MethodSM study was conducted to validate the method for detection of Listeria spp. in swab samples from stainless steel and sealed concrete surfaces. Methods and Results: In inclusivity testing, 60 of 60 Listeria spp. strains tested positive. In exclusivity testing, 31 of 31 nontarget bacterial strains tested negative. In LOD testing, the test was able to detect as few as 2 CFU of L. monocytogenes applied to a stainless steel surface. In matrix testing of inoculated stainless steel and sealed concrete surfaces, there were no statistically significant differences in method performance comparing the Listeria Right Now and U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedures as determined by probability of detection analysis. In robustness testing, modest changes to three assay operating parameters simultaneously did not significantly affect performance of the test. Conclusions and Highlights: Results can be obtained in less than 1 h using the Listeria Right Now test, allowing food industry personnel to take immediate corrective action in the case of Listeria contamination incidents.

Background: Solus One Listeria is designed to accurately detect Listeria species (Listeria grayi, L. innocua, L. ivanovii, L. marthii, L. monocytogenes, L. seeligeri, and L. welshimeri) from stainless steel and plastic environmental surface matrixes using an antibody-based technology test system paired with proprietary SOLO+ media and combined with manual or automated sample preparation method. Objective: Solus One Listeria was evaluated for inclusivity and exclusivity and a matrix comparison study fortwo environmental surfaces. Methods: Solus One Listeria was comparedwith the following reference method for the method comparison study: the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 10 from stainless steel and plastic environmental surfaces. Both the manual and automatedpreparation methods were performed for stainless steel and plastic environmental surfaces. Results: For the inclusivity and exclusivityevaluation, Solus One Listeria correctly identified all 50 target organism isolates and correctly excluded all 30 nontarget strains that were analyzed. In the method comparison study, bothSolus One Listeria manual and automated preparation methods demonstrated no significant differences based on probability of detection statistical analysis between presumptive and confirmed results or between candidate and reference methodresults for two environmental surfaces after 22–30 h of enrichment time. Probability of detection analysis of Solus One Listeria method robustness, product consistency (lot-to-lot), and stability studies using the automated preparation method demonstrated no statistically significant differences. Conclusions: The data from the study support the product claims of Solus One Listeria for the accurate detection of Listeria species, using both the manual and automated methods (using theDynex DS2 instrument), on both environmental surfaces analyzed.

Background The Clear Safety Salmonella method was modified to improve sample preparation, PCR reagents, library preparation, flow cell quality control, library loading mix, priming mix, and sequencing kit reagents and steps. Objective To evaluate the modified Clear Safety Salmonella method (manual and automated) via independent and method developer validation studies according to current AOAC INTERNATIONAL Validation Guidelines. Method Performance of the modified Clear Safety Salmonella method (manual and automated) was assessed for selectivity (using 105 inclusive and 30 exclusive strains), probability of detection in matrixes, product consistency, stability, and robustness. The modified Clear Safety Salmonella method was compared with the appropriate reference method for Salmonella detection on 4 inch × 4 inch stainless steel environmental surfaces, and in chicken carcass rinse (30 mL), raw ground chicken (375 g), dry pet food (375 g), and ready-to-eat deli turkey breast (375 g). Results The modified Clear Safety Salmonella method (manual and automated) demonstrated no statistically significant differences between the candidate and reference method probability of detection or between the presumptive and confirmed results for all target food matrixes and the stainless steel surface. Additionally, the modified method (manual and automated) detected all 105 inclusivity organisms and excluded all 30 exclusivity organisms. The product consistency and kit stability studies showed no statistical differences between lots or over the term of the kit’s shelf life. In robustness studies, changes in enrichment time, diluted sample volume, and sample volume for PCR did not show any statistical difference in terms of assay performance. Conclusions The modified Clear Safety Salmonella method (both manual and automated) is statistically equivalent to or better than the reference methods. Highlights The Clear Safety Salmonella method utilizes PCR amplification and targeted next-generation sequencing technology to selectively detect Salmonella enterica.

Background Peel PlateTM Enterobacteriaceae Bacteria (EB) is dried selective media on a 47 mm plastic plate that produces enzyme substrate colored colonies on rehydration and incubation for 24 h and up to 48 h at 37 ± 1°C. Purpose The method validation compared quantification of EB to reference methods ISO 21528:2017 Parts 1 and 2. Methods Matrixes compared were whole milk, skim powdered milk, vanilla ice cream, butter, infant formulas (soy- and dairy-based), infant cereals ± probiotic, environmental sponge swab of stainless steel surface, and poultry carcass rinse with two different peptone buffers. Results In inclusivity and exclusivity studies, the method detected 54 of 54 EB strains and did not detect 30 of 30 non-EB strains. In matrix studies, the claimed foods were tested at three contamination levels using paired analysis between the reference and Peel Plate EB methods. Colony-forming units per gram or mL [CFU/g (mL)] were log10 transformed for statistical analysis. The candidate method and reference method were shown to be equivalent by the performance requirement of all 95% confidence intervals on mean difference falling between −0.5 and +0.5 log10 CFU/g (mL). An international collaborative study with dried infant formula spiked with Cronobacter sakazakii at log10 CFU/g (mL) 1.05, 2.31, and 3.21 levels, produced method differences −0.16, 0.15, and 0.18 log10 CFU/g (mL) with repeatabilities (r) = 0.33, 0.20, and 0.12 log10 CFU/g (mL) and reproducibilities (R) = 0.45, 0.26, and 0.18 log10 CFU/g (mL). Conclusions Based on these evaluations, the candidate method is considered equivalent to the reference methods at both the 24 h and 48 h incubation periods at 37 ± 1°C. Highlights Ready to use Enterobacteriaceae method equivalent to ISO-21528:2017 Parts 1 and 2; EB test colored colonies at 37°C for 24 h are equivalent at 48 h incubation; Singlet determined CFU/mL are statistically the same as duplicate average results; EB test validated for infant formula and dairy products including with probiotics; EB test for environmental surfaces and poultry carcass rinses using peptone buffers.

Background The Solus One Salmonella immunoassay utilizes Salmonella specific selective media and automated liquid handling, for the rapid and specific detection of Salmonella species in select food types. Objective The candidate method was evaluated using 375 g test portions in an unpaired study design for a single matrix, instant non-fat dry milk (NFDM) powder. Method The matrix was compared to the United States Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 5 Salmonella reference method. Eleven participants from 10 laboratories within academia and industry, located within the United States, Mexico, South Africa, Germany, and the United Kingdom, contributed data for the collaborative study. Three levels of contamination were evaluated for each matrix: an uninoculated control level [0 colony forming units (CFU)/test portion], a low inoculum level (0.2–2 CFU/test portion) and a high inoculum level (2–5 CFU/test portion). Statistical analysis was conducted according to the Probability of Detection (POD) statistical model. Results Results obtained for the low inoculum level test portions produced a dLPOD value with a 95% confidence interval between the candidate method confirmed (both alternative and conventional confirmation procedures) and the reference method of 0.07 (−0.02, 0.15). Conclusions The dLPOD results indicate equivalence between the candidate method and the reference method for the matrix evaluated and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. False positive and false negative rates were determined for the matrix and produce values of Highlights Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis.

Background The MC-Media Pad® Rapid Aerobic Count (RAC) is a ready-to-use culture device combining a test pad coated with medium and water absorption polymers that are designed for the rapid quantification of total aerobic bacteria in food products. Objective The MC-Media Pad RAC was compared to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, Chapter 3.02: Quantitative Analysis of Bacteria in Foods as Sanitary Indicators for raw ground pork and the Standard Methods for the Examination of Dairy Products, Chapter 6: Microbial Count Methods for yogurt drink. Method The candidate method was evaluated against the reference methods using a paired study design in a multi-collaborator study, following the current AOAC INTERNATIONAL Official Methods of AnalysisSM Appendix J guidelines. Three target contamination levels (low, medium, and high) were evaluated. MC-Media Pad RAC devices were enumerated after 24 and 48 h of incubation. Results Plate counts obtained by both methods were log10-transformed and the difference of means (including 95% confidence intervals), repeatability SD, and reproducibility SD were determined for each contamination level. All 95% confidence intervals for mean difference fell easily within ±0.10, the performance requirement being ±0.5. Conclusion The MC-Media Pad RAC (for both 24 and 48 h) and both reference methods for each contamination level were therefore shown to be equivalent, with 97.5% confidence. Highlights The new method offers a convenient alternative to the reference methods for detection of aerobic plate count in food products, yielding reliable and comparable results in 24 or 48 h compared to 48 h for the reference methods.

The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for the rapid and accurate confirmation and identification of Gram-positive bacteria from select media types. This alternative method was evaluated using nonselective and selective agar plates to identify and confirm Listeria monocytogenes, Listeria species, and select Gram-positive bacteria. Results obtained by the Bruker MALDI Biotyper were compared with the traditional biochemical methods as prescribed in the appropriate reference method standards. Sixteen collaborators from 16 different laboratories located within the European Union participated in the collaborative study. A total of 36 blind-coded isolates were evaluated by each collaborator. In each set of 36 organisms, there were 16 L. monocytogenes strains, 12 non-monocytogenes Listeria species strains, and 8 additional Gram-positive exclusivity strains. After testing was completed, the total percentage of correct identifications (to both genus and species level) and confirmation from each agar type for each strain was determined at a percentage of 99.9% to the genus level and 98.8% to the species level. The results indicated that the alternative method produced equivalent results when compared with the confirmatory procedures specified by each reference method.

The iQ-Check Salmonella II Real-Time PCR test kit utilizes Salmonella-specific oligonucleotide probes and primers for the rapid and specific detection of Salmonella species in select food types. The alternative method was evaluated by using 375 g test portions in an unpaired study design for two matrices, milk chocolate and dry dog food. Each matrix was compared with the U.S. Food and Drug Administration Chapter 5 Salmonella reference method. Fourteen technicians from 12 laboratories, including academia and industry, located within the United States and Canada participated in the collaborative study. Three levels of contamination were evaluated for each matrix: an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2–2 CFU/test portion), and a high inoculum level (2–5 CFU/test portion). The statistical analysis was conducted according to the Probability of Detection (POD) statistical model. The results obtained for the low inoculum level test portions produced a difference in the candidate presumptive and confirmatory results (dLPOD) value with a 95% confidence interval of −0.05, (−0.15, 0.06) for the milk chocolate and 0.10, (−0.01, 0.21) for the dry dog food. The dLPOD results indicate an equivalence between the candidate method and reference method for the matrices evaluated, and the method demonstrated acceptable interlaboratory reproducibility as determined in the collaborative evaluation. False positive and false negative rates were determined for each matrix and produce values of

QIAGEN mericon Escherichia coli O157 Screen Plus and mericon E. coli Shiga toxin-producing E. coli (STEC) O-Type Pathogen Detection Assays use Real-Time PCR technology for the rapid, accurate detection of E. coli O157 and the “big six” (O26, O45, O103, O111, O121, O145) (non-O157 STEC) in select food types. Using a paired study design, the assays were compared with the U.S. Department of Agriculture, Food Safety Inspection Service Microbiology Laboratory Guidebook Chapter 5.09 reference method for the detection of E. coli O157:H7 in raw ground beef. Both mericon assays were evaluated using the manual and an automated DNA extraction method. Thirteen technicians from five laboratories located within the continental United States participated in the collaborative study. Three levels of contamination were evaluated. Statistical analysis was conducted according to the probability of detection (POD) statistical model. Results obtained for the low-inoculum level test portions produced a difference between laboratories POD (dLPOD) value with a 95% confidence interval of 0.00 (–0.12, 0.12) for the mericon E. coli O157 Screen Plus with manual and automated extraction and mericon E. coli STEC O-Type with manual extraction and –0.01 (–0.13, 0.10) for the mericon E. coli STEC O-Type with automated extraction. The dLPOD results indicate equivalence between the candidate methods and the reference method.

The Applied Biosystems™ RapidFinder™ STEC Detection Workflow (Thermo Fisher Scientific) is a complete protocol for the rapid qualitative detection of Escherichia coli (E. coli) O157:H7 and the “Big 6” non-O157 Shiga-like toxin-producing E. coli (STEC) serotypes (defined as serogroups: O26, O45, O103, O111, O121, and O145). The RapidFinder STEC Detection Workflow makes use of either the automated preparation of PCR-ready DNA using the Applied Biosystems PrepSEQ™ Nucleic Acid Extraction Kit in conjunction with the Applied Biosystems MagMAX™ Express 96-well magnetic particle processor or the Applied Biosystems PrepSEQ Rapid Spin kit for manual preparation of PCR-ready DNA. Two separate assays comprise the RapidFinder STEC Detection Workflow, the Applied Biosystems RapidFinder STEC Screening Assay and the Applied Biosystems RapidFinder STEC Confirmation Assay. The RapidFinder STEC Screening Assay includes primers and probes to detect the presence of stx1 (Shiga toxin 1), stx2 (Shiga toxin 2), eae (intimin), and E. coli O157 gene targets. The RapidFinder STEC Confirmation Assay includes primers and probes for the “Big 6” non-O157 STEC and E. coli O157:H7. The use of these two assays in tandem allows a user to detect accurately the presence of the “Big 6” STECs and E. coli O157:H7. The performance of the RapidFinder STEC Detection Workflow was evaluated in a method comparison study, in inclusivity and exclusivity studies, and in a robustness evaluation. The assays were compared to the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 5.09: Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products and Carcass and Environmental Sponges for raw ground beef (73% lean) and USDA/FSIS-MLG 5B.05: Detection, Isolation and Identification of Escherichia coli non-O157:H7 from Meat Products and Carcass and Environmental Sponges for raw beef trim. No statistically significant differences were observed between the reference method and the individual or combined kits forming the candidate assay using either of the DNA preparation kits (manual or automated extraction). For the inclusivity and exclusivity evaluation, the RapidFinder STEC Detection Workflow, comprising both RapidFinder STEC screening and confirmation kits, correctly identified all 50 target organism isolates and correctly excluded all 30 nontarget strains for both of the assays evaluated. The results of these studies demonstrate the sensitivity and selectivity of the RapidFinder STEC Detection Workflow for the detection of E. coli O157:H7 and the “Big 6” STEC serotypes in both raw ground beef and beef trim. The robustness testing demonstrated that minor variations in the method parameters did not impact the accuracy of the assay and highlighted the importance of following the correct incubation temperatures

Background CERTUS Environmental Listeria species Detection Kit (CERTUS EL Detection Kit) is a real-time, bio-contained assay designed to accurately detect Listeria species (L. grayi, L. innocua, L. ivanovii, L. marthii, L. monocytogenes, L. seeligeri, and L. welshimeri) from environmental surface matrixes using an antibody-coupled magnetic microparticle with a Surface Enhanced Raman Spectroscopy (SERS) nanoparticle technology test system paired with proprietary CERTUS EL Selective Growth Media and CERTUS Detection Unit. Objective The method was evaluated for AOAC®Performance Tested MethodSM certification. Methods Inclusivity and exclusivity, matrix studies, product consistency and stability were conducted to evaluate the CERTUS EL Detection Kit. Results In the matrix studies, stainless steel, ceramic tile, plastic (polystyrene) and sealed concrete environmental surfaces (4 × 4” test areas) were tested. No statistically significant differences were found by Probability of Detection analysis (POD) in any of the matrixes when results were compared to the U.S. Food and Drug Administration cultural microbiology reference method for Listeria. The CERTUS EL Detection Kit correctly identified all 50 target Listeria isolates and correctly excluded all 30 non-target strains that were analyzed. Probability of Detection analysis of CERTUS EL Detection Kit robustness, product consistency (lot-to-lot) and stability studies demonstrated no statistically significant differences, and no variation was observed between instruments. Conclusions The data collected in these studies demonstrate that the CERTUS EL Detection Kit is a reliable method for the rapid and specific detection of Listeria from stainless steel, ceramic tile, plastic (polystyrene) and sealed concrete environmental surfaces.

Background: The iQ-Check Real-Time PCR kits use PCR technology based on gene amplification and detection by a real-time PCR thermalcycler for the detection of target analytes in select food matrices. The iQ-Check E. coli O157:H7 [Performance Tested MethodSM (PTM) 020801] and STEC VirX and STEC SerO (combined PTM 121203) methods were previously validated for different matrices under different enrichment schemes. Objective: To modify the current iQ-Check E. coli O157:H7 Kit for the detection of Escherichia coli O157:H7 from 25 to 375 g for raw ground beef (17% fat), raw beef trim, and fresh spinach. In addition, a matrix extension was validated for iQ-Check E. coli O157:H7 for raw chicken breast without skin (25 g), raw chicken thigh with skin (25 g), mechanically separated chicken (25 g), and raw ground pork (25 g). The study also included the modification of the iQ-Check STEC VirX and SerO Kits for the detection of non-O157 Shiga toxin–producing E. coli (STEC) for raw ground beef (375 g), raw beef trim (375 g), and fresh spinach (375 g) from STEC Enrichment Broth to buffered peptone water (BPW). All tests were carried out at 8–22 h (10–22 h for fresh spinach). Methods: Ground beef, beef trim, and spinach were co-inoculated with E. coli O157:H7, non-O157 STECs, and Salmonella spp. and analyzed for E. coli O157:H7 and non-O157 STECs after an 8-22 h enrichment in BPW for the beef matrices and after a 10–22 h enrichment in BPW for spinach. The chicken matrices were inoculated with E. coli O157:H7 only and analyzed after an 8–22 h enrichment in BPW. The iQ-Check Free DNA Removal Solution workflow was utilized for all matrices. Confirmations at the 22 h time point and method comparisons were conducted with the appropriate reference method as outlined in the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 4A or the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapters 5.09 and 5B.05. For the iQ-Check STEC VirX and STEC SerO Kits, inclusivity and exclusivity were also performed. Results: The two inclusivity and exclusivity evaluations indicated that the test methods can accurately detect the target analytes and correctly excluded nontarget organisms after 8 h of enrichment. In the method comparison study, the iQ-Check E. coli O157:H7 and STEC VirX and STEC SerO test kits demonstrated no statistically significant differences between candidate and reference method results or between presumptive and confirmed results for all food matrices analyzed and the two time points (8 or 10 and 22 h). Both time points produced the same results, with no discrepancies. Conclusions: The iQ-Check real-time PCR kits are effective methods for the detection of E. coli O157 and non-O157 STECs (both the virulence factors and the O groups) from raw ground beef, raw beef trim, and fresh spinach in 375 g samples enriched in BPW for 8–22 h (10–22 h for fresh spinach). In addition, the iQ-Check E. coli O157 Kit is effective in detecting E. coli O157 in 25 g samples of raw chicken breast without skin, raw chicken thigh with skin, mechanically separated chicken, and raw ground pork. The iQ-Check test kits allow the end user to pair enrichments for multiple target analytes, allowing the user to prepare a single enrichment and perform a single DNA extraction. The Free DNA Removal Solution removes free DNA from samples prior to PCR analysis, protecting DNA from intact and living cells. Highlights: The method modifications were granted based on the data collected.

The Bruker MALDI Biotyper® method utilizes matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS for the rapid and accurate identification and confirmation of Gram-negative bacteria from select media types. The alternative method was evaluated using nonselective and selective agars to identify Cronobacter spp., Salmonella spp., and select Gram-negative bacteria. Results obtained by the Bruker MALDI Biotyper were compared to the traditional biochemical methods as prescribed in the appropriate reference methods. Two collaborative studies were organized, one in the United States focusing on Cronobacter spp. and other Gram-negative bacteria, and one in Europe focusing on Salmonella spp. and other Gram-negative bacteria. Fourteen collaborators from seven laboratories located within the United States participated in the first collaborative study for Cronobacter spp. Fifteen collaborators from 15 service laboratories located within Europe participated in the second collaborative study for Salmonella spp. For each target organism (either Salmonella spp. or Cronobacter spp.), a total of 24 blind-coded isolates were evaluated. In each set of 24 organisms, there were 16 inclusivity organisms (Cronobacter spp. or Salmonella spp.) and 8 exclusivity organisms (closely related non-Cronobacter spp. and non-Salmonella spp. Gram-negative organisms). After testing was completed, the total percentage of correct identifications from each agar type for each strain was determined at a percentage of 100.0% to the genus level for the Cronobacter study and a percentage of 100.0% to the genus level for the Salmonella study. For both non-Cronobacter and non-Salmonella organisms, a percentage of 100.0% was correctly identified. The results indicated that the alternative method produced equivalent results when compared to the confirmatory procedures specified by each reference method.

Background: The Polyskope 1.0 Multiplex Assay is a novel test to simultaneously detect Escherichia coli O157, non-O157 Shiga Toxin-Producing E. coli (STEC), Listeria monocytogenes, and Salmonella species in a single enrichment using real-time PCR. Objective: A Performance Tested MethodSM study was conducted to validate Polyskope 1.0 for inclusivity and exclusivity as well as a matrix comparison study. Method: This assay was evaluated in an unpaired independent validation study compared with reference methods according to AOAC INTERNATIONAL validation guidelines. Polyskope 1.0 evaluated raw ground beef (25 g), deli turkey (25 g), baby spinach (25 g), and stainless-steel environmental surface sponges (4 × 4 in. test area) after inoculation with a suspension of the three target microorganisms. All matrices were compared with appropriate reference methods from the U.S. Food and Drug Administration Bacteriological Analytical Manual, U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, or International Organization for Standardization standards. Results: Polyskope 1.0 demonstrated no statistically significant differences between candidate and reference method results or between presumptive and confirmed results for three food matrices and one environmental surface. Results from inclusivity and exclusivity evaluations indicated the test method can accurately detect the target analytes and excluded all nontarget organisms. No differences were observed with the stability or lot-to-lot evaluations. Polyskope 1.0 demonstrated robustness by remaining unaffected by small variations in method parameters, which had no statistically significant effect on the results for all eight variations. Conclusions and Highlights: Polyskope 1.0 was shown to be a specific, highly accurate, and robust method for the detection of Listeria monocytogenes, Salmonella species, non-O157 STECs, and E. coli O157 across four matrices.

Background: Listeria Right Now™ is a novel, enrichment-free test for the detection of Listeria spp. in swab samples taken from environmental surfaces. Results are available in less than 1 h. In a previous Performance Tested MethodSM (PTM) study, the test was validated for swab samples from stainless-steel and sealed concrete surfaces. Objective: A PTM matrix extension study was conducted to validate the method for the detection of Listeria spp. in swab samples from ceramic tile, plastic, and rubber surfaces. Methods: Performance of the Listeria Right Now method was compared to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual reference culture procedure for the detection of Listeria spp. in swab samples taken from inoculated ceramic tile, plastic, and rubber surfaces. Data were analyzed using a probability of detection model. Results: There were no significant differences in performance between the Listeria Right Now and reference culture methods for any of the three surfaces tested, as determined by probability of detection analysis. Conclusions: The Listeria Right Now method is an effective procedure for the detection of Listeria spp. from a variety of environmental surfaces. Highlights: Listeria Right Now provides accurate results, without enrichment, in real time. This enables food industry personnel to react swiftly to suspected Listeria contamination incidents.

Background The 3M™ Petrifilm™ Rapid E. coli/Coliform Count Plate is a selective and differential sample-ready-culture medium designed for the rapid enumeration of Escherichia coli (E. coli) and coliforms in the food and beverage industries. Objective The 3M Petrifilm Rapid E. coli/Coliform Count Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) Chapter 4 Enumeration of Escherichia coli and the Coliform Bacteria, the International Organization of Standards (ISO) 4832:2006 Microbiology of food and animal feeding stuffs — Horizontal method for the enumeration of coliforms—Colony-count technique, and ISO 16649-2:2017 Microbiology of food and animal feeding stuffs—Horizontal method for the enumeration of beta-glucuronidase-positive Escherichia coli—Part 2 Colony-count technique at 44 degrees C using bromo-4-chloro-3- indolyl beta-D-glucuronide methods for the enumeration of E. coli and coliforms in dry dog kibble. Method The candidate method was evaluated using two diluents, Butterfield's phosphate buffered diluent and peptone salt solution, in a paired study design with each reference method in a multi-laboratory collaborative study following the current AOAC Validation Guidelines. Three target contamination levels and an uninoculated control level were evaluated. Results The candidate and reference methods were not statistically different at each contamination level. Reproducibility values obtained during the collaborative study were similar between the candidate and reference methods. Conclusion These results demonstrate that the candidate method is equivalent to the reference methods. Highlight 3M Petrifilm Rapid E. coli/Coliform Count Plate was recommended for Official First Action status for enumeration of E. coli and coliforms in a broad range of foods and environmental surfaces.

Salmonella is the causative agent of many outbreaks related to spice consumption. However, because of the antimicrobial properties of various spices which hinders recovery and detection, Salmonella detection in spices remains a challenge. The objective of this study was to optimize an enrichment broth for Salmonella growth in different spices and tea, in order to maintain an adequate pH and decrease the antimicrobial effects of spices during Salmonella enrichment and subsequent detection. Salmonella contaminated spice and tea dried samples were prepared and the detection of Salmonella was assessed using the developed broth and automated DNA extraction and RT-PCR. Double strength Buffered Peptone Water (BPW) was used to maintain pH, and L-cysteine and DL-serine were added to the broth to reduce the effects of antimicrobial compounds in spices. The modified enrichment broth allowed the growth of Salmonella from each spice sample. Sample to broth ratios varied from 1:9 (garlic powder, chili peppers and tea), to 1:20 (cinnamon). The pH value of each enrichment varied but remained above 4.8. The addition of L-cysteine (30 mmol/L) allowed Salmonella recovery and growth in garlic and onion samples and the addition of DL-serine (11.23 mmol/L) allowed the recovery and growth in cinnamon. The results indicated that Salmonella detection was achieved in24 h in the modified (BPW + L-cysteine and DL-serine) enrichment broth followed by detection by RT-PCR. This protocol could allow for a more rapid, robust, and sensitive enrichment method for Salmonella in spices.

The iQ-Check Salmonella II Real-Time PCR test kit utilizes Salmonella-specific oligonucleotide probes and primers for the rapid and specific detection of Salmonella species in select food types. The alternative method was evaluated by using 375 g test portions in an unpaired study design for two matrices, milk chocolate and dry dog food. Each matrix was compared with the U.S. Food and Drug Administration Chapter 5 Salmonella reference method. Fourteen technicians from 12 laboratories, including academia and industry, located within the United States and Canada participated in the collaborative study. Three levels of contamination were evaluated for each matrix: an uninoculated control level (0 CFU/test portion), a low inoculum level (0.2-2 CFU/test portion), and a high inoculum level (2-5 CFU/test portion). The statistical analysis was conducted according to the Probability of Detection (POD) statistical model. The results obtained for the low inoculum level test portions produced a difference in the candidate presumptive and confirmatory results (dLPOD) value with a 95% confidence interval of -0.05, (-0.15, 0.06) for the milk chocolate and 0.10, (-0.01, 0.21) for the dry dog food. The dLPOD results indicate an equivalence between the candidate method and reference method for the matrices evaluated, and the method demonstrated acceptable interlaboratory reproducibility as determined in the collaborative evaluation. False positive and false negative rates were determined for each matrix and produce values of2%. Based on the data generated, the method demonstrated acceptable interlaboratory reproducibility data and statistical analysis.

QIAGEN mericon Escherichia coli O157 Screen Plus and mericon E. coli Shiga toxin-producing E. coli (STEC) O-Type Pathogen Detection Assays use Real-Time PCR technology for the rapid, accurate detection of E. coli O157 and the "big six" (O26, O45, O103, O111, O121, O145) (non-O157 STEC) in select food types. Using a paired study design, the assays were compared with the U.S. Department of Agriculture, Food Safety Inspection Service Microbiology Laboratory Guidebook Chapter 5.09 reference method for the detection of E. coli O157:H7 in raw ground beef. Both mericon assays were evaluated using the manual and an automated DNA extraction method. Thirteen technicians from five laboratories located within the continental United States participated in the collaborative study. Three levels of contamination were evaluated. Statistical analysis was conducted according to the probability of detection (POD) statistical model. Results obtained for the low-inoculum level test portions produced a difference between laboratories POD (dLPOD) value with a 95% confidence interval of 0.00 (-0.12, 0.12) for the mericon E. coli O157 Screen Plus with manual and automated extraction and mericon E. coli STEC O-Type with manual extraction and -0.01 (-0.13, 0.10) for the mericon E. coli STEC O-Type with automated extraction. The dLPOD results indicate equivalence between the candidate methods and the reference method.

The DNAble Salmonella detection assay utilizes single overnight culture enrichment, user-friendly sample preparation, and isothermal DNA amplification for Salmonella detection. This report describes studies performed in support of AOAC Research Institute Performance Tested MethodSM certification of the DNAble assay. Selectivity (inclusivity and exclusivity) studies were performed in the sponsor's laboratory. DNAble detected 119 out of 120 Salmonella isolates, representing 100 Salmonella serovars, in the inclusivity study while none of the 35 diverse non-Salmonella strains (32 species) tested was detected in the exclusivity study. Consistency (lot-to-lot and stability), instrument variation, and robustness studies were also conducted by the sponsor. Statistically equivalent assay performance was observed in these studies demonstrating robust assay manufacture and performance despite variation of multiple parameters in these challenges. Matrix studies, performed in an independent laboratory, evaluated DNAble assay performance in dry pet food, on stainless steel surfaces, and poultry environmental drag swab samples. Two sample sizes (25 and 375 g) and two culture volumes (9:1 and 3:1, v/w) were evaluated in separate matrix studies for dry pet food to provide multiple certified testing options for assay users. DNAble assay performance for dry pet food and stainless steel was compared to the procedures described in the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual, Chapter 5, Salmonella. Assay performance for drag swabs was compared to protocols dictated in the FDA Environmental Sampling and Detection of Salmonella in Poultry Houses guidelines. Matrix study results demonstrated statistically equivalent DNAble assay performance compared to these reference methods, ensuring that the DNAble assay provides results comparable to those of the reference methods.

The Applied Biosystems™ RapidFinder™ STEC Detection Workflow (Thermo Fisher Scientific) is a complete protocol for the rapid qualitative detection of Escherichia coli (E. coli) O157:H7 and the "Big 6" non-O157 Shiga-like toxin-producing E. coli (STEC) serotypes (defined as serogroups: O26, O45, O103, O111, O121, and O145). The RapidFinder STEC Detection Workflow makes use of either the automated preparation of PCR-ready DNA using the Applied Biosystems PrepSEQ™ Nucleic Acid Extraction Kit in conjunction with the Applied Biosystems MagMAX™ Express 96-well magnetic particle processor or the Applied Biosystems PrepSEQ Rapid Spin kit for manual preparation of PCR-ready DNA. Two separate assays comprise the RapidFinder STEC Detection Workflow, the Applied Biosystems RapidFinder STEC Screening Assay and the Applied Biosystems RapidFinder STEC Confirmation Assay. The RapidFinder STEC Screening Assay includes primers and probes to detect the presence of stx1 (Shiga toxin 1), stx2 (Shiga toxin 2), eae (intimin), and E. coli O157 gene targets. The RapidFinder STEC Confirmation Assay includes primers and probes for the "Big 6" non-O157 STEC and E. coli O157:H7. The use of these two assays in tandem allows a user to detect accurately the presence of the "Big 6" STECs and E. coli O157:H7. The performance of the RapidFinder STEC Detection Workflow was evaluated in a method comparison study, in inclusivity and exclusivity studies, and in a robustness evaluation. The assays were compared to the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 5.09: Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products and Carcass and Environmental Sponges for raw ground beef (73% lean) and USDA/FSIS-MLG 5B.05: Detection, Isolation and Identification of Escherichia coli non-O157:H7 from Meat Products and Carcass and Environmental Sponges for raw beef trim. No statistically significant differences were observed between the reference method and the individual or combined kits forming the candidate assay using either of the DNA preparation kits (manual or automated extraction). For the inclusivity and exclusivity evaluation, the RapidFinder STEC Detection Workflow, comprising both RapidFinder STEC screening and confirmation kits, correctly identified all 50 target organism isolates and correctly excluded all 30 nontarget strains for both of the assays evaluated. The results of these studies demonstrate the sensitivity and selectivity of the RapidFinder STEC Detection Workflow for the detection of E. coli O157:H7 and the "Big 6" STEC serotypes in both raw ground beef and beef trim. The robustness testing demonstrated that minor variations in the method parameters did not impact the accuracy of the assay and highlighted the importance of following the correct incubation temperatures.

A method modification validation study was conducted to validate the Applied Biosystems MicroSEQ™ Salmonella spp. Detection Kit for the detection of Salmonella spp. in 375 g samples of dried pet food. The MicroSEQ assay protocol, using the Applied Biosystems PrepSEQ™ Rapid Spin DNA Sample Preparation Kit, was compared to the reference method detailed in the U.S Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM; Chapter 5, Salmonella) for detection of Salmonella spp. For each method, 20 replicates were analyzed at a low contamination level of 0.2–2 CFU/test portion, five replicates were analyzed at a high level of contamination of 2–5 CFU/test portion, and five control replicates were also analyzed at 0 CFU/test portion (uninoculated). Statistical analysis was conducted using the Probability of Detection statistical test to determine the ability of the MicroSEQ Salmonella spp. Detection Kit to detect Salmonella from 375 g samples of dried pet food in comparison to the FDA-BAM reference method. The results demonstrated that the MicroSEQ Salmonella spp. Detection Kit was able to accurately detect Salmonella spp. present in dry pet food after an enrichment time of 20 h.