Evaluation of VIDAS listeria monocytogenes II (LMO2) immunoassay method for the detection of Listeria monocytogenes in foods: collaborative study

A multilaboratory study was conducted to compare the VIDAS Listeria monocytogenes II (LMO2) immunoassay and the standard cultural methods for the detection of Listeria monocytogenes in foods. Five food types-vanilla ice cream, brie cheese, cooked roast beef, frozen green beans, and frozen tilapia fish-at 3 levels were analyzed by each method. A total of 26 laboratories representing government and industry participated. In this study, 1404 test portions were analyzed of which 1152 were used in the statistical analysis. There were 448 positive by the VIDAS LMO2 assay and 457 positive by the standard culture methods. A chi2 analysis of each of the 5 food types, at the 3 inoculation levels tested, was performed. The resulting chi2 value, 0.36, indicates that overall, there are no statistical differences between the VIDAS LMO2 assay and the standard methods at the 5% level of significance.     

Evaluation of VIDAS Listeria species Xpress (LSX) immunoassay method for the detection of Listeria species in foods: collaborative study

In a multilaboratory study, the effectiveness of an alternative method for rapid screening of Listeria species compared to traditional reference methods was demonstrated in a variety of food products. A collaborative study was conducted to compare the VIDAS Listeria species Xpress (LSX) method and the standard cultural methods for the detection of Listeria species in foods. Six food types were tested: vanilla ice cream, cheddar cheese, raw ground beef, frozen green beans, deli turkey, and cooked shrimp. Each food, inoculated with a different Listeria strain at two levels and uninoculated test portions, was analyzed by each method. A total of 15 laboratories representing government and industry participated. In this study 1134 tests were analyzed in the statistical analysis. There were 490 positives by the VIDAS LSX method using the sample boiling step, 483 positives by the VIDAS LSX method using the Heat and Go system, and 439 positives by the standard culture methods. Overall, the Chi-square result for the VIDAS LSX method with boiling for all foods was 7.25, indicating a significant statistical difference between the VIDAS method and the standard methods at the 5% confidence. For the VIDAS LSX method with the Heat and Go system, the Chi-square result for all foods was 5.37, indicating a significant statistical difference between the VIDAS LSX assay with the Heat and Go system and the standard methods at the 5% level of significance. In both cases, the VIDAS method was more sensitive than the standard methods. The LSX method detects Listeria species in foods with negative or presumptive positive results in a minimum of 30 h compared to at least 5 days for the cultural methods. Based on the results of this collaborative study, it is recommended that the VIDAS LSX method be adopted as an AOAC Official Method for the detection of Listeria species in dairy products, vegetables, seafood, raw meats and poultry, and processed meats and poultry.     

VIDAS enzyme-linked immunoflourescent assay for detection of Listeria in foods: collaborative study

The VIDAS LIS method and the traditional culture methods for detection of Listeria species in food were evaluated in a multilaboratory comparative study. The 6 foods tested were either naturally contaminated or inoculated with 3 different concentrations of Listeria. Results for each food and each contamination level with the VIDAS LIS method were as good as or better than those obtained with the traditional culture method. Of 1558 samples tested, 935 were positive: 839 by the VIDAS method and 809 by standard culture methods. Overall false negative rates were 10.3 and 13.5% for the VIDAS LIS and culture methods, respectively. The false positive rate for the VIDAS LIS assay was 1.4% based on 9 VIDAS LIS positive assays that did not confirm positive by isolation of Listeria. The agreement between the VIDAS LIS and culture methods for all samples tested was 86%.     

Evaluation of VIDAS Salmonella (SLM) easy Salmonella method for the detection of Salmonella in a variety of foods: collaborative study

The VIDAS Salmonella (SLM) Easy Salmonella method is a specific enzyme-linked fluorescent immunoassay performed in the automated VIDAS instrument. The VIDAS Easy Salmonella method is a simple 2-step enrichment procedure, using pre-enrichment followed by selective enrichment in a newly formulated broth, SX2 broth. This new method was compared in a multilaboratory collaborative study to the U.S. Food and Drug Administration's Bacteriological Analytical Manual, Chapter 5 method for five food matrixes (liquid egg, vanilla ice cream, spinach, raw shrimp, and peanut butter) and the U.S. Department of Agriculture's Microbiology Laboratory Guidebook 4.04 method for deli turkey. Each food type was artificially contaminated with Salmonella at three inoculation levels. A total of 15 laboratories representing government, academia, and industry, throughout the United States, participated. In this study, 1583 samples were analyzed, of which 792 were paired replicates and 791 were unpaired replicates. Of the 792 paired replicates, 285 were positive by both the VIDAS and reference methods. Of the 791 unpaired replicates, 341 were positive by the VIDAS method and 325 were positive by the cultural reference method. A Chi-square analysis of each of the six food types was performed at the three inoculation levels tested. For all foods evaluated, the VIDAS Easy SLM method demonstrated results comparable to those of the reference methods for the detection of Salmonella.     

Evaluation of the BAX system for detection of Listeria monocytogenes in foods: collaborative study

A multilaboratory study was conducted to compare the automated BAX system and the standard cultural methods for detection of Listeria monocytogenes in foods. Six food types (frankfurters, soft cheese, smoked salmon, raw, ground beef, fresh radishes, and frozen peas) were analyzed by each method. For each food type, 3 inoculation levels were tested: high (average of 2 CFU/g), low (average of 0.2 CFU/g) and uninoculated controls. A total of 25 laboratories representing government and industry participated. Of the 2335 samples analyzed, 1109 were positive by the BAX system and 1115 were positive by the standard method. A Chi square analysis of each of the 6 food types, at the 3 inoculation levels tested, was performed. For all foods, except radishes, the BAX system performed as well as or better than the standard reference methods based on the Chi square results.     

Visual immunoprecipitate assay eight hour method for detection of enterohemorrhagic Escherichia coli O157:H7 in raw and cooked beef (modification of AOAC Official Method 996.09): collaborative study

AOAC Official Method 996.09, Visual Immunoprecipitate Assay (VIP) for Escherichia coli O157:H7, was modified to incorporate a new enrichment protocol using BioControl EHEC8 medium for testing raw and cooked beef. Foods were tested by VIP assay and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) enrichment procedure and the FDA Bacteriological Analytical Manual (BAM) isolation and confirmation techniques. A total of 15 collaborators participated. Raw and cooked ground beef were inoculated with E. coli O157:H7 at 2 different levels: a high level, where predominantly positive results were expected, and a low level where fractional recovery was anticipated. Collaborators tested 396 test portions and controls by both methods, for a total of 792 test portions. Of the 396 paired test portions, 75 were positive and 230 were negative by both the VIP and culture methods. Eleven test portions were presumptively positive by VIP and could not be confirmed culturally; 32 were negative by VIP, but confirmed positive by culture; and 65 were negative by the culture method, but confirmed positive by the VIP method. There was no statistical difference between results obtained with the VIP for EHEC 8 h method and the culture method except for cooked beef, where the VIP had significantly higher recovery for one inoculation level.     

Method extension study to validate applicability of AOAC Official Method 997.03 visual immunoprecipitate assay (VIP) for Listeria monocytogenes and related Listeria spp. from environmental surfaces: collaborative study

Test portions from 3 environmental surface types, representative of typical surfaces found in a food production facility, were analyzed by the Visual Immunoprecipitate assay (VIP) and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) culture method for Listeria monocytogenes and related Listeria species. In all cases, naturally contaminated environmental test samples were collected from an actual food production facility by sponge or swab. Test samples from concrete surfaces were collected by both swab and sponge; sponge test samples were collected from rubber surfaces, and swabs were used to sample steel surfaces. Test portions from each surface type were simultaneously analyzed by both methods. A total of 27 laboratories, representing government agencies as well as private industry in both the United States and Canada, participated in the study. During this study, a total of 615 test portions and controls was analyzed and confirmed, of which 227 were positive and 378 were negative by both methods. Nine test portions were positive by culture, but negative by the VIP. Five test portions were negative by culture, but positive by the VIP. Four test portions were negative by VIP and by culture, but confirmed positive when VIP enrichment broths were subcultured to selective agars. The data reported here indicate that the VIP method and the USDA/FSIS culture method are statistically equivalent for detection of L. monocytogenes and related Listeria species from environmental surfaces taken by sponges or swabs.     

Evaluation of the BAX system for detection of Salmonella in selected foods: collaborative study

A multilaboratory study was conducted to compare the automated BAX System to the standard cultural methods for detection of Salmonella in selected foods. Five food types--frankfurters, raw ground beef, mozzarella cheese, raw frozen tilapia fish, and orange juice--at 3 inoculation levels, were analyzed by each method. A sixth food type, raw ground chicken, was tested using 3 naturally contaminated lots. A total of 16 laboratories representing government and industry participated. In this study, 1386 samples were analyzed, of which 1188 were paired samples and 198 were unpaired samples. Of the 1188 paired samples, 461 were positive by both methods and 404 were negative by both methods. Thirty-seven samples were positive by the BAX System but negative by the standard reference method, and 11 samples were positive by standard cultural method and negative by the BAX System. Of the 198 unpaired samples, 106 were positive by the BAX System and 60 were positive by the standard cultural method. A Chi square analysis of each of the 6 food types, at the 3 inoculation levels tested, was performed. For all foods, the BAX System demonstrated results comparable to those of the standard reference methods based on the Chi square results.     

Validation of a microwell DNA probe assay for detection of Listeria spp. in foods Performance-tested method 010403

A new DNA hybridization assay in microwell format for detection of Listeria spp. in foods and environmental samples was developed. This assay uses Listeria-specific oligonucleotide probes labeled with horseradish peroxidase and a photometrically determined end point. Validation studies with 15 different food commodities and a variety of environmental sample types were conducted to compare the performance of this alternative test versus reference methods. Meats, seafood, dairy products, and vegetables comprised the categories of food tested. Food samples were inoculated at 2 levels and refrigerated or frozen for at least 72 h. Uninoculated (negative) control samples were included in each trial. Samples were enriched according to the procedure recommended by either the U.S. Food and Drug Administration (FDA) or the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS). Samples enriched for 24 h were transferred to Oxford agar plates and incubated for 24 h. The surface of the plates was then swabbed and any growth present was transferred to phosphate buffer solution for the performance of the DNA assay. A standard confirmation procedure was used to compare the number of positive samples obtained with the DNA method versus reference methods. Statistical analyses of the results indicate that the proposed alternative method performs equally to cultural reference methods. The DNA assay is able to detect as low as 1 colony-forming unit of Listeria in a 25 g food sample, with results available as early as 48 h after the start of sample enrichment.     

Assurance enzyme immunoassay eight hour method for detection of enterohemorrhagic Escherichia coli O157:H7 in raw and cooked beef (modification of AOAC Official Method 996.10): collaborative study

AOAC Official Method 996.10, Assurance Enzyme Immunoassay (EIA) for Escherichia coli O157:H7 (EHEC), was modified to incorporate a new enrichment protocol using BioControl EHEC8 medium for testing raw and cooked beef. Foods were tested by EIA and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) enrichment conditions and the FDA Bacteriological Analytical Manual (BAM) isolation and confirmation techniques. A total of 14 collaborators participated. Raw and cooked ground beef were inoculated with E. coli O157:H7 at 2 different levels: a high level where predominantly positive results were expected, and a low level where fractional recovery was anticipated. Collaborators tested 378 test portions and controls by both the 8 h EIA and the USDA/FSIS enrichment methods, for a total of 756 test portions. Of the 378 paired test portions, 75 were positive and 212 were negative by both methods. Thirteen test portions were presumptively positive by EIA and could not be confirmed culturally; 30 were negative by EIA, but confirmed positive by culture; and 65 were negative by the culture method, but confirmed positive by the EIA method. There was no statistical difference between results obtained with the Assurance EIA for EHEC 8 h method and the culture method for raw ground beef. The Assurance EIA had a significantly higher recovery for cooked beef.     

Method extension study to validate applicability of AOAC Official Method 996.14 Assurance polyclonal enzyme immunoassay for detection of Listeria monocytogenes and related Listeria spp. from environmental surfaces: collaborative study

Test portions from 3 environmental surface types, representative of typical surfaces found in a food production facility, were analyzed by the Assurance Listeria Polyclonal Enzyme Immunoassay (EIA) and the U.S. Department of Agriculture/Food Safety and Inspection Service (USDA/FSIS) culture method for Listeria monocytogenes and related Listeria species. In all cases, naturally contaminated environmental test samples were collected from an actual food production facility by sponge or swab. Test samples from concrete surfaces were collected by both swab and sponge; sponge test samples were collected from rubber surfaces, and swabs were used to sample steel surfaces. Test portions from each surface type were simultaneously analyzed by both methods. A total of 23 collaborators, representing government agencies, as well as private industry in both the United States and Canada, participated in the study. During this study, a total of 550 test portions and controls was analyzed and confirmed, of which 207 were positive and 336 were negative by both methods. Six test portions were positive by culture, but negative by the EIA. Three test portions were negative by culture, but positive by the EIA. Two test portions were negative by EIA and by culture, but confirmed positive when EIA enrichment broths were subcultured to selective agars. The data reported here indicate that the Assurance Listeria EIA method and the USDA/FSIS culture method are statistically equivalent for detection of L. monocytogenes and related Listeria species from environmental surfaces taken by sponges or swabs.     

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.     

3M Petrifilm Staph Express Count plate method for the enumeration of Staphylococcus aureus in selected types of processed and prepared foods: collaborative study

The 3M Petrifilm Staph Express Count plate method was compared with AOAC Official Method 975.55 for the enumeration of Staphylococcus aureus in selected foods. Five foods--frozen lasagna, custard, frozen mixed vegetables, frozen hashbrowns, and frozen batter-coated mushrooms--were analyzed for S. aureus by 13 collaborating laboratories. For each food tested, the collaborators received 8 blind test samples consisting of a control sample, a low inoculation level, a medium inoculation level, and a medium inoculation level with background flora, each in duplicate. The mean log10 counts for the methods were comparable for all 5 foods. The repeatability and reproducibility variances of the 24 h Petrifilm Staph Express Count plate method were similar to those of the 72 h standard method.     

Rapid determination of Listeria monocytogenes by automated enzyme-linked immunoassay and nonradioactive DNA probe

A rapid and reliable analytical method was developed to detect and confirm the presence of Listeria monocytogenes in raw and partially processed foods. Forty-nine food samples (25 mixed cut vegetable salad, 12 smoked salmon, and 12 sterile smoked salmon) were individually inoculated with high levels [10-100 colony forming units (cfu)/25 g sample] and low levels (1-10 cfu/25 g sample) of L. monocytogenes, and were screened using the Vitek Immuno Diagnostic Assay (VIDAS) Listeria monocytogenes (VIDAS LMO)]. Positive test results were confirmed as L. monocytogenes by nonradioactive DNA probe. All samples inoculated with high levels of L. monocytogenes were detected by VIDAS and 96% were confirmed as L. monocytogenes by DNA probe. VIDAS LMO detected 89% of samples inoculated with low levels of L. monocytogenes, and 87% of these were confirmed as positive by DNA probe. In addition, 12 other samples (4 from each of mixed cut vegetable salad, smoked salmon, and sterile smoked salmon) were inoculated with high levels of L. ivanovii, L. seeligeri, L. welshimeri, L. innocua, L. grayi, and L. murrayi. Samples were assayed by the same protocol and all gave negative results. Compared with the cultural method, the VIDAS LMO nonradioactive DNA probe combination is highly specific, discriminates between L. monocytogenes and all other Listeria species, and reduces analytical time.     

Modification of enrichment protocols for TECRA Listeria Visual Immunoassay method 995.22: collaborative study

A collaborative study was conducted to validate new enrichment methods for the TECRA Listeria Visual Immunoassay (TLVIA). These new methods incorporate a newly formulated medium, TECRA Listeria Enrichrment Broth, which does not contain the highly toxic antifungal agent, cycloheximide. The new procedures will provide an alternative to the enrichment procedures described in AOAC Method 995.22. Three food types (raw ground beef, lettuce, and ice cream) were analyzed in the United States, and 2 food types (cooked turkey and cooked fish fillets) were analyzed in Australasia. Thirty collaborators participated in the study, 16 in Australasia and 14 in the United States. With the exception of one batch of ground beef, comparison of the proportion of positive test portions (p > or = 0.05) showed no significant difference between the TLVIA and the reference method for the 5 foods at 3 inoculation levels. For the one batch of naturally contaminated raw ground beef, the TLVIA gave significantly more confirmed positive results than the reference method.     

Evaluation of VIDAS Salmonella (SLM) immunoassay method with Rappaport-Vassiliadis (RV) medium for detection of Salmonella in foods: collaborative study

A collaborative study was conducted to compare the VIDAS Salmonella (SLM) with Rappaport-Vassiliadis (RV) method for detection of Salmonella in foods to the current standard method presented in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) and the culture method presented in AOAC's Official Methods of Analysis. The VIDAS SLM with RV method uses tetrathionate broth in combination with RV medium in place of selenite cystine broth for selective enrichment, thereby eliminating the hazardous waste issue for laboratories. Twenty five laboratories participated in the evaluation, each testing one or more of 8 test products: nonfat dry milk, dried egg, soy flour, lactic casein, milk chocolate, raw ground pork, raw ground turkey, and raw peeled shrimp. Results of the study showed no significant differences in the numbers of confirmed positive samples with the VIDAS SLM with RV procedure and the BAM/AOAC culture procedure. The VIDAS SLM with RV method was effective for rapid detection of Salmonella in foods. It is recommended that AOAC INTERNATIONAL modify the VIDAS Salmonella SLM procedure to include the RV method.     

Evaluation of the VIDAS staph enterotoxin II (SET 2) immunoassay method for the detection of staphylococcal enterotoxins in selected foods: collaborative study

A multilaboratory study was conducted to determine the limit of detection (LOD) of Staphylococcal enterotoxins (SET) in 5 foods. Cooked chicken, ham, potato salad, pasteurized liquid whole milk, and canned mushrooms were each spiked with a different enterotoxin (A, B, C1, D, or E), and tested at 0.25 and 0.5 ng/g SET levels to determine the LOD of the assay for those foods in a collaborative study. Unspiked controls were also included. A total of 19 laboratories representing government and industry participated. In this study, 1674 test portions were analyzed, of which 1638 were used in the statistical analysis. Of the 1638 test portions used in the statistical analysis, 1104 were spiked test portions, of which 1073 were positive by the VIDAS Staph enterotoxin II (SET 2) method. The detection rates at the 0.25 ng/mL level were cooked chicken, 98.2%; ham, 99.0%; potato salad, 99.1%; liquid whole milk, 85.2%; and canned mushrooms, 100%. The detection rates at the 0.5 ng/mL level were cooked chicken, 97.4%; ham, 98.1%; potato salad, 100%; liquid whole milk, 99.0%; and canned mushrooms, 100%. The data indicate that the SET 2 method is capable of detecting SET at 0.25 ng/g in cooked chicken, ham, potato salad, and canned mushrooms and at 0.5 ng/g in pasteurized liquid whole milk.     

Dry rehydratable film method for rapid enumeration of coliforms in foods (3M Petrifilm Rapid Coliform Count plate): collaborative study

A rehydratable dry-film plating method for coliforms in foods, the 3M Petrifilm Rapid Coliform Count plate method, was compared with the U.S. Food and Drug Administration's Bacteriological Analytical Manual method for nondairy foods and the American Public Health Association's Standard Methods for the Examination of Dairy Products (SMEDP) method for dairy foods. Six food types, vanilla ice cream, cheddar cheese, fresh refrigerated uncooked pasta, wheat flour, prepared frozen macaroni and cheese, and frozen hash browns, were analyzed for coliforms by 11 collaborating laboratories. For each food product tested, the collaborators received 8 blind samples consisting of a control sample and 3 levels of inoculated sample, each in duplicate. The mean log counts for the methods were comparable. The repeatability and reproducibility variances of the Petrifilm Rapid Coliform Count method at 14 and 24 h were not significantly different from those of the standard methods.     

Validation study to demonstrate the equivalence of a minor modification (TECRA ULTIMA protocol) to AOAC Method 998.09 (TECRA Salmonella Visual Immunoassay) with the cultural reference method

The TECRA Salmonella Visual Immunoassay (VIA) using Rappaport-Vassiliadis RV[R10] as a single selective enrichment broth has Final Action approval (AOAC Method 998.09). TECRA has recently developed a protocol (TECRA ULTIMA), which involves the addition of a new additive to a 1 mL aliquot of the RV[R10] broth, prior to the heat-killing step, thereby allowing the RV[R10] broth to be tested directly in the kit and thus eliminating the need for the 2 h post-enrichment in M broth. An in-house validation study was conducted to compare the modified AOAC Method 998.09 to the reference culture method. Three foods were used in the study: Naturally contaminated raw ground poultry at high (10-50 cells/25 g), and low (1-5 cells/25 g) levels; and milk powder and peanut butter, artificially inoculated at low and high levels with Salmonella bovismorbificans and S. enterica Mbandaka, respectively. Twenty test portions were analyzed for each level with 10 uninoculated control samples per food. Overall, no significant differences (p <0.05) were observed when the proportion of positive test portions for the modified VIA were compared with that for the reference method. This minor modification, which employs the additive (provided in the TECRA ULTIMA SALMONELLA Test Kit) to permit the direct analysis of RV[R10] broth has demonstrated the utility of the TECRA ULTIMA SALMONELLA protocol. It is recommended that the minor modification to Method 998.09 be approved First Action as an additional option within the method.     

Salmonella in foods: new enrichment procedure for TECRA Salmonella Visual Immunoassay using a single rv(R10) only, TT only, or dual rv(R10) and TT selective enrichment broths (AOAC official method 998.09): collaborative study

A collaborative study was conducted to compare a new enrichment procedure for the TECRA Salmonella Visual Immunoassay (TSVIA) with the reference method given in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (7th Ed.). Three food types (milk powder, pepper, and soy flour) were analyzed in Australia and 3 food types (milk chocolate, dried egg, and raw turkey) were analyzed in the United States. Thirty-eight collaborators participated in the study. The TECRA method was evaluated using both Rappaport-Vassiliadis R10 (RV(R10)) and tetrathionate (TT) broths for selective enrichment. M broth cultures arising from each of the 2 selective enrichment broths were tested in the TSVIA using 2 individual wells, one for each selective broth, and a single well to test the pooled selective enrichment broths. The results for the pooled enrichment broths were reported elsewhere. This study presents the results for the use of single enrichment broths, i.e., RV(R10) only or TT only, with the TSVIA. No significant differences (p > 0.05) were observed for the pairwise comparison of the proportion of positive samples for either RV(R10) or TT used as a single enrichment broth for the TSVIA with that for the reference method.