Microbiology Laboratory Guidebook Research Articles

Independent evaluation of a DNA microarray system for Salmonella detection in ground beef

A new DNA microarray test kit has been developed to detect foodborne pathogens in various food matrices. This study focuses on evaluating the PathogenDx microarray-based system to detect Salmonella in ground beef and verify critical parameters that could interfere with the method's effectiveness, such as enrichment incubation time, ground beef fat content, inclusivity, exclusivity, and analytical sensitivity. Sample preparation protocols were evaluated at 6, 8, 12, 18, and 24 h enrichment times at varying bacterial levels to identify optimal conditions to detect the invA gene using the PathogenDx microarray. An 8 h enrichment step was selected based on 100% detection when initial inoculum levels were ≥5 CFU/g, and fractional detection was achieved when the concentration was as low as 1 CFU/g. Thus, the detection of Salmonella using the PathogenDx microarray system can be conducted in 12.5 h, including sample preparation, labeling PCR, hybridization, and analysis. Regarding fat content, there was no significant difference in detection rates of PathogenDx protocol among the highest and lowest commercially sold lean-to-fat ratios of ground beef. Inclusivity and exclusivity experiments showed that Salmonella was correctly identified 100% of the time. Using the ground beef matrix, PathogenDx method is comparable to the United States Department of Agriculture's Microbiology Laboratory Guidebook methodology for detection, which correctly identified Salmonella in 100% of the samples. Salmonella was detected between 93.33 and 100% when ground beef was inoculated with 1 and 5 CFU/g, respectively.

Prevalence and Antibiotic Resistance of Salmonella and Campylobacter Isolates from Raw Chicken Breasts in Retail Markets in the United States and Comparison to Data from the Plant Level

Chicken is the most popular meat in the United States, and consumers may be exposed to multidrug resistant Salmonella and Campylobacter through consumption of retail chicken breasts. This study aimed to (i) determine the percentage of raw, packaged, retail chicken breasts from 27 metro areas that tested positive for Salmonella and Campylobacter; (ii) investigate the antibiotic susceptibility profiles of a subset of the isolates; and (iii) compare the Salmonella prevalence data to establishment level Salmonella categorization data published by the U.S. Department of Agriculture (USDA). USDA Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) methodology was used to isolate and identify Salmonella (n = 672), Campylobacter (n = 499) from 400 g samples. National Antimicrobial Resistance Monitoring System (NARMS) methodology was followed for antimicrobial susceptibility testing of Salmonella (n = 52) and Campylobacter (n = 16) isolates. Salmonella was found in 8.6% of samples and Campylobacter in 4.2%. Having a 3 rating in USDA's Salmonella Categorization of Individual Establishments for chicken parts was predictive of having a higher Salmonella percent positive in our data set (p ≤ 0.05). A total of 73.1% of Salmonella isolates, and 62.5% of Campylobacter isolates were resistant to ≥one class of antibiotics, with 48.1% of Salmonella isolates resistant to ≥three classes. Current results support interventions that take a 'farm-to-fork' approach with distinction by poultry types and parts as well as serovars, to lower antibiotic resistant Salmonella infections in humans due to poultry. Highlights:Salmonella was found in 8.6% and Campylobacter in 4.2% of chicken breasts tested; A 3 rating by USDA was predictive of a higher Salmonella percent positive; 48.1% of Salmonella isolates were resistant to 3 or more classes of antibiotics.

Assessment of Reference Method Selective Broth and Plating Media with 19 Listeria Species Highlights the Importance of Including Diverse Species in Listeria Method Evaluations

Reference methods developed for detection of Listeria monocytogenes are commonly used for detection of Listeria at the genus level. Improved method performance data are needed because this genus has expanded from 6 to 26 species and now includes several Listeria sensu lato species, which can have phenotypes distinct from those of Listeria sensu stricto. We evaluated growth of 19 Listeria species, including 12 recently described Listeria sensu lato species, using the media specified by (i) the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual, (ii) the U.S. Department of Agriculture (USDA) Microbiology Laboratory Guidebook, and (iii) the International Organization for Standardization (ISO). The FDA broth enrichment procedure allowed all species to grow to detectable levels (≥4 log CFU/mL), yielded the highest mean growth (7.58 log CFU/mL), and was the only procedure with which no Listeria sensu lato species yielded significantly higher growth than did a comparison Listeria sensu stricto species. With the USDA and ISO broth enrichment procedures, several Listeria sensu lato species yielded significantly higher growth than did either Listeria seeligeri or Listeria ivanovii, suggesting that these two Listeria sensu stricto species could be outgrown by Listeria sensu lato species. On selective and differential agar media, L. seeligeri, L. ivanovii, and Listeria grayi produced colonies with atypical morphology and/or growth of these species was inhibited (which may lead to incorrect classification of a sample as negative), whereas several newly described Listeria sensu lato species grew to high levels and produced colonies with typical morphology. Overall, our study results indicate that the ability to detect various Listeria species can be impacted by the specific broth and selective and differential agar used. Our data can help guide selection of appropriate media and detection methods for environmental Listeria monitoring programs and methods that are most likely to detect the targeted Listeria groups (e.g., Listeria sensu stricto, which appear to be the most appropriate index organisms for the pathogen L. monocytogenes).

Validation of the 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) for the Detection of Shiga Toxin Gene (stx1 and/or stx2) in Additional Matrixes: AOAC Performance Tested MethodSM 071903.

BackgroundThe 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) method is based on gene amplification by the use of real-time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) from Shiga toxin-producing enterohemorrhagic Escherichia coli (STEC) in enriched foods.ObjectiveThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method was evaluated as a Level 2 method modification to add new matrixes to the certified claim: 25 g fresh raw ground beef (approximately 75% lean), 375 g raw beef trim (approximately 75% lean), 375 g fresh raw ground pork (approximately 70% lean), 375 g fresh raw poultry parts, and 25 g sprouts.MethodsMatrix studies were conducted to assess the method’s performance compared to the US Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, 5C.00 for meat and poultry, and to the US Food and Drug Administration Bacteriological Analytical Manual, Ch. 4A for sprouts, using an unpaired study design.ResultsThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method demonstrated no significant differences between presumptive and confirmed results or between candidate and reference method results for any of the matrices tested.Conclusion and HighlightsThe data collected in these studies demonstrate that the 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) is a reliable method for the rapid and specific detection of STEC in fresh raw ground beef (approximately 75% lean), fresh raw beef trim (approximately 75% lean), fresh raw ground pork (approximately 70% lean), fresh raw poultry parts, and sprouts.

Validation of the 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) for the Detection of Shiga Toxin Gene (stx1 and/or stx2) and Intimin Gene (eae): AOAC Performance Tested MethodSM 071902.

BackgroundThe 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) method is based on gene amplification by the use of real-time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) and intimin gene (eae) from Shiga toxin-producing enterohemorrhagic Escherichia coli (STEC) in enriched foods.ObjectiveThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) method was evaluated as a Level 2 method modification to add new matrixes to the certified claim: 25 g fresh raw ground beef (approximately 75% lean), 375 g fresh raw ground pork (approximately 70% lean), 375 g fresh raw poultry parts, and 25 g sprouts.MethodsMatrix studies were conducted to assess the method’s performance compared to the U. S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, 5C.00 for meat and poultry, and to the U. S. Food and Drug Administration Bacteriological Analytical Manual, Ch. 4A for sprouts, using an unpaired study design.ResultsThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) method demonstrated no significant differences between presumptive and confirmed results or between candidate and reference method results for any of the matrixes tested.Conclusion and HighlightsThe data collected in these studies demonstrate that the 3M Molecular Detection Assay 2 - STEC Gene Screen (stx and eae) is a reliable method for the rapid and specific detection of STEC in fresh raw ground beef (approximately 75% lean), fresh raw ground pork (approximately 70% lean), fresh raw poultry parts, and sprouts.

Withdrawn: Investigation on Salmonella enterica, Escherichia coli, and coliforms in beef from Ethiopian abattoirs: A potential risk of meat safety.

The aims of this study were to determine the prevalence of Salmonella enterica (S. enterica) antimicrobial resistance based upon phenotypic assessment, and level of sanitation indicator organisms from 150 beef carcasses collected from three representative abattoirs in eastern, central, and southern Ethiopia. Samples were screened for S. enterica prevalence following the U.S. Department of Agriculture Microbiology Laboratory Guidebook (MLG) and confirmed by real‐time PCR. The S. enterica isolates were phenotypically evaluated for susceptibility to a panel of 13 antimicrobials using disk diffusion method as described in Clinical and Laboratory Standard Institutes guidelines. In addition, the coliform, total coliform, and generic E. coli populations were quantified by plating onto E. coli/coliform petrifilms. A total of 45 isolates of S. enterica were confirmed using real‐time PCR. The overall prevalence of S. enterica at the carcass level was 22.7% (95% CI, 16.0–30.0). However, based on the sampling points, S. enterica prevalence detected on hides was 13.3% (95% CI, 8.0–18.7), pre‐evisceration was 12.0% (95% CI, 7.3–17.3), and post‐evisceration was 4.7% (95% CI, 1.3–8.0). The prevalence of S. enterica was significantly lower on post‐evisceration as compared to hides (p = .009) and pre‐evisceration (p = .022). No differences were detected for the prevalence of S. enterica between abattoirs (p = .346). Finally, the prevalence of S. enterica was highest during the wet season (p = .011). The overall mean log CFU/cm2 ± SD of generic E. coli, coliform, and total coliform counts were 4.55 ± 0.99, 4.91 ± 1.13, and 4.98 ± 1.09, respectively. About 20% of S. enterica exhibited phenotypic multidrug resistant. The most frequently detected resistance was to Tetracycline (28.9%), followed by Streptomycin (22.2%) and Sulfisoxazole (20.0%). The data from this study highlight the need to implement interventions on improvement of sanitary practices in abattoirs.

Validation of the 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) for the Detection of Shiga Toxin Gene (stx1 and/or stx2) in Fresh Raw Ground Beef and Fresh Spinach: AOAC Performance Tested MethodSM 071903.

BackgroundThe 3M™ Molecular Detection Assay 2 - STEC Gene Screen (stx) method is based on gene amplification by the use of real time loop-mediated isothermal amplification when used with the 3M Molecular Detection System for the rapid and specific detection of Shiga toxin gene (stx1 and/or stx2) from Shiga toxin-producing Escherichia coli (STEC) in enriched foods. The stx assay does not differentiate between stx1 and stx2 but detects the presence of stx1 and/or stx2.ObjectiveThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method was evaluated for AOAC®  Performance Tested MethodsSM certification.MethodsMatrix studies, inclusivity/exclusivity, robustness testing, product stability, and lot-to-lot variability testing were conducted to assess the method’s performance.ResultsThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) demonstrated equivalent results to the United States Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook Chapter 5C.00 reference method for fresh raw ground beef, and the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 4A reference method for fresh spinach. The 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) detected all STEC E. coli strains (E. coli strains with stx1 and/or stx2 genes) and did not detect any of the 45 strains from the exclusivity panel. Robustness testing indicated that small variations in critical test parameters did not adversely affect the assay’s performance. Product consistency and stability testing demonstrated no differences between the lots evaluated.ConclusionThe data collected in these studies demonstrate that the 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) is a reliable method for the rapid and specific detection of Shiga toxin-producing E. coli in raw ground beef and spinach.HighlightsThe 3M Molecular Detection Assay 2 - STEC Gene Screen (stx) method is suitable for the rapid and specific detection of Shiga toxin-producing E. coli in fresh raw ground beef, and spinach.

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.

BackgroundThe 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.ObjectiveThe 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.MethodIn 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.ResultsIn 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.ConclusionsBased on the data generated, the method demonstrated acceptable interlaboratory reproducibility data and statistical analysis.HighlightsDue 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.

Comparative Evaluation of the SMARTCHECK® Listeria monocytogenes Detection Kit for GENECHECKER® UF-300 Real-Time PCR System for Select Foods: AOAC Performance Tested MethodSM 042101.

The candidate method, SMARTCHEK® Listeria monocytogenes Detection Kit, is designed for rapid detection of L. monocytogenes in select food matrixes, for use in conjunction with the Genesystems Rapi:chipTM and GENECHECKER®UF-300 real-time PCR system. The independent laboratory evaluation consisted of an inclusivity and exclusivity study, product consistency study, robustness study, stability study, and a matrix study. The paired matrix study tested stainless-steel environmental sponges, uncured ready-to-eat deli turkey, ready-to-eat deli ham, cooked shrimp, and fresh bagged spinach. The robustness study evaluated the method by changing three parameters. The accelerated stability and product consistency study was conducted at 8, 16, 24, 32, and 40 days. The candidate method was compared to the US Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, 8.11 Isolation and Identification of Listeria monocytogenes from Red Meat, Poultry, Ready-to-Eat Siluriformes (Fish) and Egg Products, and Environmental Samples and to the US Food and Drug Administration Bacteriological Analytical Manual Chapter 10: Detection of Listeria monocytogenes in foods and Environmental Samples, and Enumeration of Listeria monocytogenes in Foods following a paired study design. All 50 L. monocytogenes isolates were detected in all three broths tested for inclusivity, while all 37 non-L. monocytogenes isolates were correctly excluded. In the matrix study, the candidate method showed no significant differences between confirmed candidate method results and reference method results for all food matrixes evaluated. The candidate method is an effective method for the qualitative detection of L. monocytogenes in select food matrixes. The SMARTCHECK Listeria monocytogenes Detection Kit assay allows for fast, reliable detection of L. monocytogenes with results obtained in approximately 35 min after 18-48 h pre-enrichment, depending on the matrix.

Comparative Evaluation of the SMARTCHEK Salmonella Species Detection Kit for GENECHECKER UF-300 Real-Time PCR System with Rapi:chipTM and Rapi:DirectTM Lysis Buffer for Select Foods: AOAC Performance Tested MethodSM 032101

The candidate method, SMARTCHEK Salmonella spp. Detection Kit assay is designed for rapid detection of Salmonella spp. in select food matrixes, for use in conjunction with the Genesystems Rapi:chipTM and GENECHECKER UF-300 real-time PCR system. The independent laboratory evaluation consisted of an inclusivity and exclusivity study, product consistency study, robustness study, stability study, and a matrix study. The paired matrix study tested romaine lettuce, peanut butter, liquid whole egg, chicken carcass rinsate, and raw chicken. The robustness study evaluated the method by changing three method parameters. The accelerated stability and product consistency study was conducted at 8, 16, 24, 32, and 40 days. The candidate method was compared to the U.S. 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 and to the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 5: Salmonella following a paired study design. All 108 Salmonella isolates were detected in all three broths tested for inclusivity, while all 35 non-Salmonella isolates were correctly excluded. In the matrix study, the candidate method showed no significant differences between confirmed candidate method results and reference method results for all food matrixes evaluated. The candidate method is an effective method for the qualitative detection of Salmonella spp. in select food matrixes. The SMARTCHEK Salmonella spp. Detection Kit assay allows for fast, reliable detection of Salmonella spp. with results obtained after 24 h of incubation and a PCR run time of only 35 min.

Occurrence of Listeria monocytogenes in Counter-Sliced Turkey Meat Samples from Independent Delis in New York City

Research suggests that small, independent delicatessens are less likely to follow proper sanitation procedures, including slicer inspection, which could lead to a higher likelihood of these delis being a reservoir for Listeria monocytogenes growth and cross-contamination. This study was undertaken to determine the incidence of L. monocytogenes in counter-sliced turkey deli meat obtained from independent delis in an urban city. Deli meat, counter-sliced on site, was collected from 118 independent delis in New York City. The samples were analyzed for L. monocytogenes using the U.S. Department of Agriculture Microbiology Laboratory Guidebook methodology for isolation and confirmation. The selection criteria for delis included using the city's restaurant inspection and grading system. Two samples, from separate delis, were confirmed as positive for L. monocytogenes (1.69%). Analysis of the genomic sequences of one of the samples revealed a close match to a cluster of six clinical isolates, which were part of an ongoing multistate listeriosis outbreak spanning four states. The sequence of the second isolate matched a clinical isolate in a neighboring state. Both isolates were obtained from delis that did not have the top inspection grade. Although a snapshot of one urban area, this study is the first report on the current incidence of L. monocytogenes on counter-sliced deli meat from independent deli establishments. This study suggests that these delis can potentially serve as sources of L. monocytogenes contamination or contribute to downstream foodborne listeriosis. Information provided by city inspection and grading systems, in addition to the letter grade, may serve as a tool to identify delis with potential L. monocytogenes contamination issues and a basis for product and environmental sampling by public health authorities.

Evaluation of the GENE-UP®Salmonella Method for the Detection of Salmonella Species in a Broad Range of Foods and Select Environmental Surfaces: Collaborative Study, First Action 2020.02.

The GENE-UP®Salmonella assay (Performance Tested MethodSM [PTM] 121802) is a PCR detection method that utilizes fluorescence resonance energy transfer (FRET) hybridization probes for the rapid detection of Salmonella species in foods and on environmental surfaces. The purpose of this validation was to evaluate the method's interlaboratory performance for submission to AOAC INTERNATIONAL for adoption as First Action Official Method of AnalysisSM. The GENE-UP method was evaluated in a multi-laboratory study using unpaired test portions for one food matrix, raw ground beef (80% lean). The candidate method was compared to the US Department of Agriculture (USDA), Food Safety Inspection Service, Microbiology Laboratory Guidebook (MLG) 4.10 reference method. An alternative confirmation procedure, using ASAP™ and CHROMID®Salmonella chromogenic agars, was included in the validation study. Fifteen collaborators from 14 laboratories participated. Three levels of contamination were evaluated: a non-inoculated control level (0 CFU/test portion), a low contamination level (∼0.7 CFU/test portion), and a high contamination level (∼2 CFU/test portion). Data were analyzed using the probability of detection (POD) statistical model. The dLPODC values with 95% confidence interval for the GENE-UP Salmonella method, with either alternative or traditional confirmation, were: 0.00 (-0.03, 0.03), -0.02 (-0.15, 0.12), and 0.02 (-0.03, 0.09) for the non-inoculated, low, and high contamination levels respectively. The dLPODC results demonstrate no difference in performance between the candidate method and reference method for the matrix evaluated. The GENE-UP Salmonella method, with ASAP and CHROMID Salmonella, provides industry with a simplified, rapid, and accurate workflow for the detection of Salmonella in a broad range of foods and select environmental surfaces.

Validation of PhageDx™Salmonella Assay in Raw Ground Turkey and Powdered Infant Formula: AOAC Performance Tested MethodSM 121904.

BackgroundThe PhageDx™Salmonella Assay is based on the infection of Salmonella spp. by specific bacteriophages and expression of a luciferase reporter gene. Results are generated in as little as 9.5 h for raw ground turkey and 18.5 h for milk-based powdered infant formula (PIF).ObjectiveAn AOAC Performance Tested MethodsSM (PTM) study was conducted to validate the PhageDx Salmonella Assay for the detection of Salmonella in 25 g raw ground turkey and 100 g PIF test portions.MethodThe performance of the PhageDx Salmonella Assay was compared to that of the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 4.10 for raw ground turkey and the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 for PIF. Inclusivity/exclusivity, product consistency and stability, and robustness testing were conducted.ResultsThere was no significant difference between the 25 g raw ground turkey and 100 g PIF PhageDx Salmonella Assay and the USDA/FSIS MLG 4.10 and FDA/BAM Chapter 5, respectively. The reporter bacteriophages were specific for Salmonella and infected 108 strains in inclusivity testing. They did not infect 30 non-Salmonella bacteria in exclusivity testing. Robustness testing showed that the method performed well with specific deviations from the standard protocol. Consistency and stability testing demonstrated that the recombinant phage gave consistent results across three production lots and was stable when stored under appropriate conditions for at least eight months.ConclusionsThe data collected in the validation study demonstrate that the PhageDx Salmonella Assay meets the qualifications for PTM status. HighlightsThe PhageDx Salmonella Assay is a rapid, specific, sensitive assay capable of detecting a wide range of Salmonella spp. with a significantly shorter turn around time than the USDA/FSIS and FDA reference methods.

PhageDx™ E. coli O157:H7 Assay in Ground Beef Level 3 Modification to Add 375 g Beef Trim and New Method Procedure for 25 g Ground Beef: AOAC Performance Tested MethodSM 081601.

BackgroundThePhageDx™ E. coli O157:H7 Assay originally required a 5 h enrichment period and a 10 mL test sample for 25 g ground beef. The proposed method modifications seek to include beef trim (375 g) matrix and a new method procedure for 25 g ground beef comprised of 6–7 h enrichment and 1 mL test sample.ObjectiveTo validate the method modifications for PhageDx E. coli O157:H7 Assay to include beef trim (375 g) matrix and modify enrichment time and test sample size for 25 g ground beef.MethodFor both matrixes, pre-warmed tryptic soya broth (TSB; 42 ± 1°C) was added in a 3:1 (media:sample size) ratio, blended, and enriched for 9–10 h (beef trim, 375 g) or 6 h (ground beef, 25 g) at 42 ± 1°C. One milliliter samples were transferred to a microfuge tube, centrifuged, and the supernatant removed. The pellet was resuspended in 0.2 mL of TSB and phage reagent was added. Samples were incubated for 2 h at 37 ± 1°C. After infection, the sample was centrifuged, and 150 µl of the supernatant was transferred to a 96-well plate. Then, lysis buffer and luciferase substrate were added and the sample read on a luminometer to determine the presence or absence of E. coli O157:H7.ResultsNo significant differences were found between the PhageDx E. coli O157:H7 Assay method modifications and the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) reference method.ConclusionsThe independent study demonstrated that the PhageDx E. coli O157:H7 Assay method modifications meet the qualifications for Performance Tested Method SM status.HighlightsThe PhageDx E. coli O157:H7 Assay is a rapid detection method capable of detecting E. coli O157:H7 in 25 g ground beef and 375 g beef trim samples in as little as 8.5 h and 11.5 h total turn around time, respectively.

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.

BackgroundOne 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.ObjectiveThe purpose of the validation study was to measure the effectiveness of the OBOP Salmonella method in comparison to reference culture procedures.MethodPerformance 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.ResultsIn 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.ConclusionsResults 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.HighlightsThe 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 MC-Media Pad® Rapid Aerobic CountDevice for the Enumeration of Total Aerobic Counts in a Variety of Foods: Collaborative Study, FirstAction Official MethodSM 2019.02.

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. 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. 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. 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. 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. 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.

Modification of the Bio-Rad iQ-Check Listeria spp. Kit for the Detection of Listeria Species in Environmental Surfaces.

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. 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. 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. 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. 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. The method modification was granted based on the data collected.

Modification and Matrix Extension of the Bio-Rad iQ-Check E. coli O157:H7, STEC VirX, and STEC SerO Test Kits for the Detection of Shiga Toxin-Producing Escherichia coli (STEC) and Escherichia coli O157 From a Single Enrichment.

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. 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). 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. 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. 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. The method modifications were granted based on the data collected.

Independent Validation for the Polyskope 1.0 Multiplex Pathogen Detection Assay for the Detection of Shiga Toxin-Producing Escherichia coli Non-O157 STEC, Escherichia coli O157, Listeria monocytogenes, and Salmonella Species.

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. A Performance Tested MethodSM study was conducted to validate Polyskope 1.0 for inclusivity and exclusivity as well as a matrix comparison study. 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. 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.

Independent Validation for the Polyskope 1.0 Multiplex Pathogen Detection Assay for the Detection of Shiga Toxin-Producing Escherichia coli Non-O157 STEC, Escherichia coli O157, Listeria monocytogenes, and Salmonella Species

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.