A comparison of the BAX system method to the U.S. Food and Drug Administration's Bacteriological Analytical Manual and International Organization for Standardization reference methods for the detection of Salmonella in a variety of soy ingredients.

The performances of two DuPont BAX System PCR assays for detecting Salmonella on a variety of low-moisture soy ingredients were evaluated against the U. S. Food and Drug Administration's Bacteriological Analytical Manual (FDA BAM) method or the International Organization for Standardization (ISO) 6579 reference method. These evaluations were conducted as a single laboratory validation at an ISO 17025 accredited third-party laboratory. Validations were conducted on five soy ingredients: isolated soy protein (ISP), soy fiber, fluid soy lecithin, deoiled soy lecithin, and soy nuggets, using a paired-study design. The ISP was analyzed as both 25- and 375-g composite test portions, whereas all other sample matrices were analyzed as 375-g composite test portions. To evaluate 25-g test portions of ISP, the test material was inoculated using Salmonella enterica subsp. enterica serovar Mbandaka (Q Laboratories isolate 11031.1). Salmonella enterica subsp. enterica serovar Tennessee (Q Laboratories isolate 11031.3) was used for all other trials. For each trial of the method comparison, 25 samples were analyzed for each matrix: 5 uninoculated controls and 20 samples inoculated at low levels (0.2 to 2 CFU per test portion) that were targeted to achieve fractionally positive results (25 to 75%). Using McNemar's chi-square analysis, no significant difference at P ≥ 0.05 (χ(2) ≤ 3.84) was observed between the number of positives obtained by the BAX System and the reference methods for all five test matrices evaluated. These studies indicate that the BAX System PCR assays, in combination with the single buffered peptone water primary enrichment and subsequent brain heart infusion regrowth step, demonstrate equivalent sensitivity and robustness compared with the FDA BAM and ISO reference methods for both 25- and 375-g composite samples. Moreover, there was no observed reduction of sensitivity in the larger 375-g composite samples for all five matrices.

Detection of Salmonella species in a variety of foods by the DuPont Bax system real-time PCR assay for Salmonella: first action 2013.02.

A multilaboratory study was conducted to evaluate the ability of the DuPont BAX System Real-Time PCR Assay for Salmonella to detect the target species in a variety of foods and environmental surfaces. Internal validation studies were performed by DuPont Nutrition & Health on 24 different sample types to demonstrate the reliability of the test method among a wide variety of sample types. Two of these matrixes-pork and turkey frankfurters and pasteurized, not-from-concentrate orange juice without pulp-were each evaluated in 14 independent laboratories as part of the collaborative study to demonstrate repeatability and reproducibility of the internal laboratory results independent of the end user. Frankfurter samples were evaluated against the U.S. Department of Agriculture, Food Safety and Inspection Service reference method as a paired study, while orange juice samples were evaluated against the U.S. Food and Drug Administration reference method as an unpaired study, using a proprietary media for the test method. Samples tested in this study were artificially inoculated with a Salmonella strain at levels expected to produce low (0.2-2.0 CFU/test portion) or high (5 CFU/test portion) spike levels on the day of analysis. For each matrix, the collaborative study failed to show a statistically significant difference between the candidate method and the reference method using the probability of detection statistical model.

Modification of the BAX Salmonella test kit to include a hot start functionality (modification of AOAC Official Method 2003.09).

In 2010, the BAX System PCR assay for Salmonella was modified to include a hot start functionality designed to keep the reaction enzyme inactive until PCR begins. To validate the assay's Official Methods of Analysis status to include this procedure modification, an evaluation was conducted on four food types that were simultaneously analyzed with the BAX System and either the U.S. Food and Drug Administration's Bacteriological Analytical Manual or the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook reference method for detecting Salmonella. Identical performance between the BAX System method and the reference methods was observed. Additionally, lysates were analyzed using both the BAX System Classic and BAX System Q7 instruments with identical results using both platforms for all samples tested. Of the 100 samples analyzed, 34 samples were positive for both the BAX System and reference methods, and 66 samples were negative by both the BAX System and reference methods, demonstrating 100% correlation. No instrument platform variation was observed. Additional inclusivity and exclusivity testing using the modified test kit demonstrated the test kit to be 100% accurate in evaluation of test panels of 352 Salmonella strains and 46 non-Salmonella strains.

DuPont Qualicon BAX System assay for genus Listeria 24E.

The new BAX System PCR Assay for Genus Listeria 24E was evaluated for detecting Listeria spp. in frankfurters, spinach, cooked shrimp, queso fresco cheese, and on stainless steel surfaces with a single-stage enrichment in BAX System 24 Listeria Enrichment Broth (24 LEB). Method comparison studies performed on samples with low-level inoculates showed that the BAX System demonstrates a sensitivity equivalent or superior to the U.S. Food and Drug Administration's Bacteriological Analytical Manual and the U.S. Department of Agriculture-Food Safety and Inspection Service culture methods, but with a significantly shorter time to result. Tests to evaluate inclusivity and exclusivity returned no false-negative and no false-positive results on a diverse panel of isolates, and tests for lot-to-lot variability and tablet stability demonstrated consistent performance. Ruggedness studies determined that none of the factors examined, within the range of deviations from specified parameters examined, affect the performance of the assay.

Evaluation of DuPont Qualicon Bax System PCR assay for yeast and mold.

Evaluations were conducted to test the performance of the BAX System PCR assay which was certified as Performance Tested Method 010902 for screening yeast and mold in yogurt, corn starch, and milk-based powdered infant formula. Method comparison studies performed on samples with low-level inoculates showed that the BAX System demonstrates a sensitivity equivalent to the U.S. Food and Drug Administration's Bacteriological Analytical Manual culture method, but with a significantly shorter time to obtain results. Tests to evaluate inclusivity and exclusivity returned no false-negative and no false-positive results on a diverse panel of isolates, and tests for lot-to-lot variability and tablet stability demonstrated consistent performance. Ruggedness studies determined that none of the factors examined affected the performance of the assay.

DuPont Qualicon BAX System polymerase chain reaction assay. Performance Tested Method 100201.

A recent outbreak of Salmonella in peanut butter has highlighted the need for validation of rapid detection methods. A multilaboratory study for detecting Salmonella in peanut butter was conducted as part of the AOAC Research Institute Emergency Response Validation program for methods that detect outbreak threats to food safety. Three sites tested spiked samples from the same master mix according to the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) method and the BAX System method. Salmonella Typhimurium (ATCC 14028) was grown in brain heart infusion for 24 h at 37 degrees C, then diluted to appropriate levels for sample inoculation. Master samples of peanut butter were spiked at high and low target levels, mixed, and allowed to equilibrate at room temperature for 2 weeks. Spike levels were low [1.08 most probable number (MPN)/25 g]; high (11.5 MPN/25 g) and unspiked to serve as negative controls. Each master sample was divided into 25 g portions and coded to blind the samples. Twenty portions of each spiked master sample and five portions of the unspiked sample were tested at each site. At each testing site, samples were blended in 25 g portions with 225 mL prewarmed lactose broth until thoroughly homogenized, then allowed to remain at room temperature for 55-65 min. Samples were adjusted to a pH of 6.8 +/- 0.2, if necessary, and incubated for 22-26 h at 35 degrees C. Across the three reporting laboratories, the BAX System detected Salmonella in 10/60 low-spike samples and 58/60 high-spike samples. The reference FDA-BAM method yielded positive results for 11/60 low-spike and 58/60 high-spike samples. Neither method demonstrated positive results for any of the 15 unspiked samples.

Testing of swine feces obtained through the National Animal Health Monitoring System's Swine 2000 study for the presence of Escherichia coli O157:H7.

Fecal samples collected from healthy pigs from 13 of the top 17 swine-producing states were tested for Escherichia coli O157:H7 as part of the National Animal Health Monitoring System Swine 2000 study. Serogroup O157 strains were isolated from 106 of 2,526 fecal samples. None of the isolates were positive by PCR for the fliCh7 (H7 flagellin) gene or for the hly933 (hemolysin) gene; however, one isolate was positive for the stxl gene (Shiga toxin 1), an additional four isolates were positive for the stx2 gene (Shiga toxin 2), and three isolates possessed the eae gene (intimin).

Relatedness of Listeria monocytogenes Isolates recovered from selected ready-to-eat foods and listeriosis patients in the United States.

Pulsed-field gel electrophoresis and serotyping were performed for 544 isolates of Listeria monocytogenes, including 502 isolates recovered from contaminated samples from 31,705 retail ready-to-eat (RTE) food products and 42 isolates recovered from human cases of listeriosis. The isolates were from Maryland (294 isolates) and California (250 isolates) and were collected in 2000 and 2001. The isolates were placed into 16 AscI pulsogroups (level of relatedness within each group, > or =66%), 139 AscI pulsotypes (levels of relatedness, > or =25% to 100%), and eight serotypes (serotypes 1/2a, 1/2b, 1/2c, 3a, 3b, 4b, 4c, and 4d). The most frequently found pulsotypes belonged to either pulsogroup A (150 food isolates plus 4 clinical isolates) or pulsogroup B (104 food isolates plus 5 clinical isolates). The majority of the 502 food isolates were either serotype 1/2a (298 isolates) or serotype 1/2b (133 isolates), whereas the majority of the 42 clinical isolates were either serotype 1/2a (19 isolates) or serotype 4b (15 isolates). Additionally, 13 clinical isolates displayed pulsotypes also found in food isolates, whereas the remaining 29 clinical isolates displayed 24 unique pulsotypes. These data indicate that most (86%) of the L. monocytogenes subtypes found in the RTE foods sampled belonged to only two serotypes and that 90% of the isolates displayed 73 pulsotypes, with 107 isolates displaying pulsotype 1. These data should help define the distribution and relatedness of isolates found in RTE foods in comparison with isolates that cause listeriosis.

Validation of the USDA/ARS package rinse method for recovery of Listeria monocytogenes from naturally contaminated, commercially prepared frankfurters.

The utility of the U.S. Department of Agriculture/Agricultural Research Service (USDA/ARS) package rinse method for recovering Listeria monocytogenes from the surface of contaminated foods was validated in comparison to the standard USDA/Food Safety and Inspection Service (FSIS) product composite enrichment method and two other methods using frankfurters from a lot with a known package prevalence rate of approximately 16% for this pathogen. One hundred packages from this batch of naturally contaminated, commercially prepared frankfurters were examined as follows: (i) the package exudative fluid was removed and tested using the standard USDA/FSIS product composite enrichment method; (ii) approximately 5 to 7 portions of frankfurters were removed to obtain a 25-g composite of meat that was then processed using the standard USDA/FSIS product composite enrichment method: (iii) 50 ml of 0.1% peptone water was added to each package, and the USDA/ARS package rinse method was performed on the remaining contents; and (iv) after removing the rinse fluid, the solid contents remaining in each package were directly enriched using the USDA/FSIS product composite enrichment method. These four methods identified that 7, 6, 15, and 9 of the 100 packages tested positive for the pathogen, respectively. Although no single approach yielded a positive result for every package that tested positive for L. monocytogenes by any one of the four sampling strategies, the USDA/ARS package rinse method was appreciably (P < 0.05) better than either the package exudate enrichment method or the standard USDA/FSIS product composite enrichment method at recovering the bacterium. These findings validate the sensitivity and ease of use of the USDA/ARS package rinse method using naturally contaminated frankfurters and argue strongly for its adoption for routine screening of ready-to-eat products that are prone to surface contamination with undesirable microbes such as L. monocytogenes.

Use of pulsed-field gel electrophoresis to characterize the heterogeneity and clonality of salmonella isolates obtained from the carcasses and feces of swine at slaughter.

Salmonella enterica isolates were recovered from swine at a collaborating processing plant over a 2-month period in the spring of 2000. In the present study, molecular subtyping by pulsed-field gel electrophoresis (PFGE) was performed on the 581 confirmed Salmonella isolates from the 84 Salmonella-positive samples obtained from the previous study. A total of 32 different PFGE pulsotypes were observed visually, and a BioNumerics software analysis clustered those pulsotypes into 12 PFGE groups. The B, F, and G groups predominated throughout the sampling period and were isolated from 39, 22, and 13% of the swine, respectively. In addition, multiple isolates were obtained from 67 of the 84 Salmonella-positive samples, and subtyping revealed multiple PFGE profiles in 35 of these 67 (62%) samples. Both carcass and fecal isolates of Salmonella were recovered from 13 swine, resulting in "matched" samples. Molecular typing of the 252 isolates recovered from the matched samples revealed that 7 (54%) of the 13 carcasses were contaminated with Salmonella pulsotypes that were not isolated from the feces of the same animal. Conversely, from 6 of the 13 (46%) matched animals, Salmonella clonal types were isolated from the feces that were not isolated from the carcass of the same animal. These data establish that each lot of swine introduces new contaminants into the plant environment and that swine feces from one animal can contaminate many carcasses. In addition, these results indicate that the examination of multiple Salmonella isolates from positive samples is necessary to determine the variety of potential contaminants of swine carcasses during slaughter and processing.

Recovery rate of Listeria monocytogenes from commercially prepared frankfurters during extended refrigerated storage.

To assess the prevalence of Listeria monocytogenes in vacuum-sealed packages of frankfurters, about 33,000 packages (1 lb each) were obtained by a third-party contractor from 12 volunteer commercial manufacturers over a 2-year period. The 12 producers, each of which contributed about 2,700 packages of frankfurters from one production run, comprised 9 large and 3 small plants located in eight U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) districts in 10 states. Five days after manufacture, 500 packages were sampled at the USDA/Agricultural Research Service (ARS) Eastern Regional Research Center (ERRC) in Wyndmoor, Pa., by the USDA/ARS package rinse method. At regular intervals during subsequent storage at 4 and 10 degrees C, an additional 200 packages were tested for the pathogen at each sampling point. From a statistical perspective, L. monocytogenes was not recovered from any of the products of nine of the producers, whereas the pathogen was recovered at rates of 1.5% (plant 367), 2.2% (plant 439), and 16% (plant 133) from the products of the remaining three plants. In total, 532 of 32,800 (1.6%) packages of frankfurters tested positive for the pathogen. The recovery rates did not change appreciably over time, there was no appreciable difference in L. monocytogenes recovery rates with respect to frankfurter storage temperature (4 or 10 degrees C), and the seasonality of manufacture had no influence on recovery rate. Molecular subtyping of multiple L. monocytogenes-positive isolates from each plant revealed that profile A (serotype 1/2a) was displayed by about 90% of the 1,105 isolates tested. However, in some cases it was also possible to recover more than one profile from a given plant. This study provides estimates of the prevalence, types, and viability of L. monocytogenes associated with commercially prepared frankfurters during extended refrigerated storage.

Isolation of Escherichia coli O157:H7 from intact colon fecal samples of swine.

Escherichia coli O157:H7 was recovered from colon fecal samples of pigs. Polymerase chain reaction confirmed two genotypes: isolates harboring the eaeA, stx(1), and stx(2) genes and isolates harboring the eaeA, stx(1), and hly(933) genes. We demonstrate that swine in the United States can harbor potentially pathogenic E. coli O157:H7.

Recovery of Listeria monocytogenes from vacuum-sealed packages of frankfurters: comparison of the U.S. Department of Agriculture (USDA) food safety and inspection service product composite enrichment method, the USDA Agricultural Research Service (ARS) product composite rinse method, and the USDA-ARS package rinse method.

This study compared three methods for the recovery of Listeria monocytogenes from commercially prepared and vacuum-packaged frankfurters that were inoculated with a five-strain mixture of this pathogen at averages of 22 and 20,133 CFU per package over three trials. The presence and levels of the pathogen were determined by (i) the U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) product composite enrichment method, involving the selective enrichment of a 25-g composite of product and the subsequent plating of this product onto selective agar plates; (ii) the USDA Agricultural Research Service (ARS) product composite rinse method, involving the rinsing of a 25-g composite of product with 0.1% peptone water and the subsequent plating of a portion of the rinse fluid directly onto selective agar plates; and (iii) the USDA-ARS package rinse method, involving the use of 25 ml of 0.1% peptone water to rinse the entire contents of a package and the subsequent plating of a portion of the rinse fluid directly onto selective agar plates. For packages inoculated with 20,133 CFU. L. monocytogenes was recovered at a frequency (percentage of packages positive) of 100% by each of the three methods. The pathogen was recovered at efficiencies (percentages of recovery of L. monocytogenes) of 43 and 94% with the USDA-ARS product rinse method and the USDA-ARS package rinse method, respectively. For packages inoculated with 22 CFU, L. monocytogenes was recovered at frequencies of 17, 10, and 100% by the USDA-FSIS product composite enrichment method, the USDA-ARS product composite rinse method, and the USDA-ARS package rinse method, respectively. The pathogen was recovered at efficiencies of 20 and 95% with the USDA-ARS product composite rinse method and the USDA-ARS package rinse method, respectively. In a related study, the USDA-ARS package rinse method was the only method that detected the pathogen in 60 packages from each of five brands of frankfurters purchased from local grocery stores. These data establish that the USDA-ARS package rinse method is markedly more sensitive, as well as demonstrably more rapid and facile, than either the approved USDA-FSIS product composite enrichment method or the USDA-ARS product composite rinse method in determining the presence or absence of L. monocytogenes and establishing the levels of the pathogen that may be on the surface of ready-to-eat foods such as frankfurters.