An Alternative Rapid Confirmation Method for Identifying Listeria monocytogenes from a Variety of 125 g Food Samples Within Two Days of a PCR Presumptive Positive.

Cultural confirmation following detection of a Listeria monocytogenespresumptive positive can take 3-7 days to finalize; this uncertainty is a point of frustration for food producers needing to make time-sensitive disposition decisions. To address the demand for shortened time-to-results, an alternative L. monocytogenes confirmation method consisting of two components, (i) a secondary screen using a different rapid method, and (ii) concurrent cultural isolation followed by next-day colony identification was evaluated. For the study, four food matrices (hot dogs, peanut butter, frozen vegetables, and multicomponent frozen meals) were inoculated with low levels (0.36-1.39 MPN/125 g) of L. monocytogenes per the AOAC guidelines for a matrix study. Analyses were performed on 125 g test portions and started with a PCR primary screen (Bio-Rad iQ-Check Listeria monocytogenes II). Next, all enriched food samples underwent a secondary screen by bioMérieux's GENE-UP LMO2 Real-Time PCR and VIDAS LMX ELFA along with streaking onto RAPID'L.mono Agar. Presumptive positive L. monocytogenes colonies were identified utilizing a high throughput rapid identification method (Hygiena's BAX System L. monocytogenes Real-Time PCR assay, Neogen's ANSR isothermal nucleic acid amplification assay, and Bruker's MALDI Biotyper). Importantly, this study evaluated multiple commercially available options for the secondary screen (n = 2) and rapid identification (n = 3) to allow for easy adoption by testing laboratories. Overall, there was no statistically significant difference (p ≤ 0.05) between the number of L. monocytogenes-positive 125 g samples obtained by the cultural reference method and the alternative confirmation methods (regardless of which method combinations were evaluated). Additionally, this study supports that, when both the primary and secondary screen methods yield a positive result, the sample could be considered a confirmed positive for L. monocytogenes.

Cooption of regulatory modules for tektin paralogs during ciliary band formation in a marine annelid larva.

Tektins are a highly conserved family of coiled-coil domain containing proteins known to play a role in structure, stability and function of cilia and flagella. Tektin proteins are thought to form filaments which run the length of the axoneme along the inner surface of the A tubule of each microtubule doublet. Phylogenetic analyses suggest that the tektin family arose via duplications from a single tektin gene in a unicellular organism giving rise to four and five tektin genes in bilaterians and in spiralians, respectively. Although tektins are found in most metazoans, little is known about their expression and function outside of a handful of model species. Here we present the first comprehensive study of tektin family gene expression in any animal system, in the spiralian annelid Platynereis dumerilii. This indirect developing species retains a full ancient spiralian complement of five tektin genes. We show that all five tektins are expressed almost exclusively in known ciliary structures following the expression of the motile cilia master regulator foxJ1. The three older bilaterian tektin-1, tektin-2, and tektin-4 genes, show a high degree of spatial and temporal co-regulation, while the spiralian specific tektin-3/5A and tektin-3/5B show a delay in onset of expression in every ciliary structure. In addition, tektin-3/5B transcripts show a restricted subcellular localization to the most apical region near the multiciliary arrays. The exact recapitulation of the sequence of expression and localization of the five tektins at different times during larval development indicates the cooption of a fixed regulatory and cellular program during the formation of each ciliary band and multiciliated cell type in this spiralian.

Validation of MICA Legionella for Enumeration of Legionella pneumophila in Sanitary Waters and Cooling Tower Waters: AOAC Performance Tested MethodSM 032201.

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.

Validation of the AquaCHROM™ ECC for the Detection and Enumeration of Coliforms and Escherichia coli in Water Samples: AOAC Performance Tested MethodSM 072202.

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.

Validation of the Assurance® GDS for Cronobacter Tq II in Infant Formulas, Infant Cereals, Ingredients, and Environmental Samples Collaborative Study: First Action 2021.08.

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 <2%. 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.

Evaluation of the Thermo Scientific SureTectTM Listeria Species PCR Assay in a Broad Range of Foods and Selected Environmental Surfaces: Pre-Collaborative and Collaborative Study, First Action 2021.06.

BACKGROUND: 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. OBJECTIVE: 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. METHODS: 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. 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 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. CONCLUSION: Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis. HIGHLIGHTS: 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.

Independent Study for the Detectx Combined Assay for the Detection of Aspergillus, Salmonella, and STEC (stx1 and/or 2) in Dried Cannabis Flower and Dried Hemp Flower: Level 3 Modification Study 012201.

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.

Evaluation of the Thermo ScientificTM SureTectTMListeria monocytogenes PCR Assay in a Broad Range of Foods and Selected Environmental Surfaces: Pre-Collaborative and Collaborative Study, First Action 2021.05.

BACKGROUND: 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. OBJECTIVE: 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. 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. 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 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. CONCLUSIONS: Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis. HIGHLIGHTS: 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.

Validation of the Modified Clear Safety Salmonella for Detection of Salmonella enterica in Selected Poultry and Pet Food Matrixes and on Stainless Steel: AOAC Performance Tested MethodSM 111802.

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.

Evaluation of the Thermo Scientific SureTect Salmonella Species PCR Assay in a Broad Range of Foods and Select Environmental Surfaces: Pre-Collaborative and Collaborative Study: First Action 2021.02.

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 <2%. The two PCR thermocyclers (QS5, 7500 Fast) performed equivalently. There were no result differences between candidate method confirmations and reference method confirmations. In the pre-collaborative matrix extension, the results from the matrix studies showed a comparable performance between the candidate method and the tested reference methods. 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.

Validation of the Clear Safety Listeria Method for Detection of Listeria Species in Hot Dogs and on Environmental Surface Matrixes: AOAC Performance Tested MethodSM 091901.

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.

Validation of the Thermo Scientific SureTect™ Escherichia coli O157:H7 and STEC Screening PCR Assay and SureTect™ Escherichia coli STEC Identification PCR Assay for the Detection of Escherichia coli O157:H7 and the Escherichia 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: AOAC Performance Tested MethodSM 012102.

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.

Validation of the Thermo Scientific™ SureTect™Staphylococcus aureus PCR Assay for the Detection of Staphylococcus aureus in Dairy Matrixes: AOAC Performance Tested MethodSM 052101.

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.

Validation of the Thermo Scientific SureTect™ Campylobacter jejuni, C. coli, and C. lari PCR Kit for the Detection of Campylobacter jejuni, C. coli, and C. lari in Raw Poultry and Ready-to-Cook Poultry Products: AOAC Performance Tested MethodSM 012101.

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.

Validation of the One Broth One Plate for Salmonella Method for Detection of Salmonella Spp. in Select Food and Environmental Samples: AOAC Performance Tested MethodSM 102002.

BACKGROUND: One Broth One Plate for Salmonella (OBOP Salmonella) is a rapid and simple method for detection of Salmonella spp. in food and environmental samples using traditional culture methodology. The method utilizes single-step enrichment followed by plating to a selective/differential, chromogenic agar. OBJECTIVE: The purpose of the validation study was to measure the effectiveness of the OBOP Salmonella method in comparison to reference culture procedures. METHOD: Performance of the OBOP Salmonella method was compared to that of the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5 reference method for queso fresco, smoked salmon, cantaloupe, chocolate, black pepper, chili powder, dry pet food, and sponge samples from a stainless steel surface, or to that of the U.S. Department of Agriculture Microbiology Laboratory Guidebook Chapter 4.10 method for raw ground turkey, chicken carcass rinse, and pasteurized liquid egg. Inclusivity/exclusivity, robustness, and stability/lot-to-lot consistency testing was also performed. RESULTS: In the matrix study, there were no statistically significant differences in performance between the OBOP Salmonella and reference methods, as determined by probability of detection analysis (P < 0.05), for any of the matrixes examined. All 104 Salmonella spp. strains produced positive results in inclusivity testing, and all 33 non-salmonellae exclusivity strains tested negative with the OBOP Salmonella method. CONCLUSIONS: Results of the validation study show that the OBOP Salmonella method is a reliable procedure for detection of Salmonella spp. in select matrixes. The method is simple to perform, requires no specialized equipment, and produces results in as little as 37 h. HIGHLIGHTS: The OBOP Salmonella method was awarded AOAC PTMSM (#102002) for detection of Salmonella in queso fresco, smoked salmon, cantaloupe, chocolate, black pepper, chili powder, dry pet food, sponge samples on a stainless steel surface, raw ground turkey, chicken carcass rinse, and pasteurized liquid egg. The method is also approved by MicroVal® for a broad range of foods under certification number 2019LR88.

Evaluation of the 3M™ Petrifilm™ Rapid E. coli/Coliform Count Plate for the Enumeration of E. coli and Coliforms: Collaborative Study, First Action: 2018.13.

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.

Validation of the Reveal® 3-D for Peanut Lateral Flow Test: AOAC Performance Tested MethodSM 111901.

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.

Evaluation of the CERTUS Environmental Listeria Species Detection Kit for the Detection of Listeria Species on Environmental Surfaces: AOAC Performance Tested MethodSM 101802.

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.

Evaluation of the MC-Media Pad® Rapid Aerobic Count Device for the Enumeration of Total Aerobic Counts in a Variety of Foods: Collaborative Study, First Action Official MethodSM 2019.02.

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.

BL30SEC Method for Detection of Beta-Lactams in Raw Commingled Cow Milk: AOAC Performance Tested MethodSM 061902.

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.