Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 999.09 [Visual Immunoprecipitate (VIP) for Salmonella] to the reference culture methods

The Visual Immunoprecipitate (VIP) for the Detection of Salmonella in Foods, AOAC Official Method 999.09, has been modified to use a simplified housing for the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Three foods were analyzed. In total, valid results were obtained from 125 samples and controls. Results showed that the modified VIP for Salmonella is equivalent to the reference culture methods for the detection of Salmonella.     

Validation study to demonstrate the equivalence of a minor modification of Assurance Enzyme Immunoassay method, Official Method 996.14, for detection of Listeria in foods and environmental surfaces to the reference culture methods

The Assurance Enzyme Immunoassay (EIA) for the Detection of Listeria in Foods and Environmental Surfaces, AOAC Official Method 996.14, has been modified to combine the separate antibody and conjugate addition steps into one. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture methods. Two food matrixes and one environmental surface were analyzed. In total, there were valid results from 145 samples and controls. Results showed that the Assurance EIA for Listeria spp. is equivalent to the reference culture methods for the detection of Listeria.     

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 996.09 [Visual Immunoprecipitate (VIP) for E. coli O157:H7] to the reference culture methods

The Visual Immunoprecipitate (VIP) for the Detection of E. coli O157:H7 in Foods, AOAC Official Method 996.09, has been modified to use a simplified plastic housing for the device. A methods comparison study was conducted to demonstrate the equivalence of this modification to the reference culture method. Three foods were analyzed. In total, valid results were obtained from 240 samples and controls. Results showed that the VIP for E. coli O157:H7 is equivalent to the reference culture methods for the detection of E. coli O157:H7.     

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.     

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.     

SDIX RapidChek Listeria F.A.S.T. environmental test system for the detection of Listeria species on environmental surfaces

The SDIX RapidChek Listeria F.A.S.T. test system was validated against the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) cultural reference method for the detection of Listeria species on stainless steel, plastic, rubber, and painted concrete. The SDIX method uses a proprietary RapidChek Listeria enrichment media for a one-step, 24-40 h enrichment at 30 degrees C, and detects Listeria on an immunochromatographic lateral flow device in 10 min. Different Listeria species were used to spike each of the environmental surfaces. Environmental surfaces were spiked at levels ranging from 50 to 400 CFU/surface (1 in.2 swabs for painted concrete, 4 in.(2) for sponge). A total of 120 spiked samples were tested by the SDIX method at 24 and 40 h and the cultural reference method. Total confirmed positives were 49, 54, and 48 for the SDIX 24 h method, the SDIX 40 h method, and the USDA-FSIS cultural reference method, respectively. Nonspiked samples from all environmental surfaces were reported as negative for Listeria spp. by all methods. The overall Chi square was 0.017 (P = 0.104) and 0.611 (P= 0.566) after a 24 and 40 h enrichment, respectively, indicating that the test method was equivalent in performance to the reference method at both enrichment times. The SDIX method was evaluated for the detection of 50 Listeria and 35 non-Listeria bacterial strains. All 50 Listeria strains were detected by the method (100% sensitivity). Five out of 35 non-Listeria species gave light test signals when grown in nonselective broth culture and tested undiluted. However, when grown in the RapidChek Listeria F.A.S.T. proprietary media, only one bacterial strain (Staphylococcus aureus) was detected, giving a very low test signal (97% specificity). The method was shown to be robust toward several alterations in testing and storage conditions.     

Reveal Listeria 2.0 test for detection of Listeria spp. in foods and environmental samples

A Performance Tested Method validation study was conducted for a new lateral flow immunoassay (Reveal Listeria 2.0) for detection of Listeria spp. in foods and environmental samples. Results of inclusivity testing showed that the test detects all species of Listeria, with the exception of L. grayi. In exclusivity testing conducted under nonselective growth conditions, all non-listeriae tested produced negative Reveal assay results, except for three strains of Lactobacillus spp. However, these lactobacilli are inhibited by the selective Listeria Enrichment Single Step broth enrichment medium used with the Reveal method. Six foods were tested in parallel by the Reveal method and the U.S. Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) reference culture procedure. Considering data from both internal and independent laboratory trials, overall sensitivity of the Reveal method relative to that of the FDA/BAM procedure was 101%. Four foods were tested in parallel by the Reveal method and the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) reference culture procedure. Overall sensitivity of the Reveal method relative to that of the USDA-FSIS procedure was 98.2%. There were no statistically significant differences in the number of positives obtained by the Reveal and reference culture procedures in any food trials. In testing of swab or sponge samples from four types of environmental surfaces, sensitivity of Reveal relative to that of the USDA-FSIS reference culture procedure was 127%. For two surface types, differences in the number of positives obtained by the Reveal and reference methods were statistically significant, with more positives by the Reveal method in both cases. Specificity of the Reveal assay was 100%, as there were no unconfirmed positive results obtained in any phase of the testing. Results of ruggedness experiments showed that the Reveal assay is tolerant of modest deviations in test sample volume and device incubation time.     

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.     

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 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.     

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC methods 996.09, Vip for EHEC and 996.10, assurance Eia EHEC with the reference culture method for the detection of Escherichia coli O157:H7 in beef

The Visual Immunoprecipitate Assay (VIP) method for the detection of enterohemorrhagic Escherichia coli O157:H7 (VIP for EHEC) and Assurance Enzyme Immunoassay (EIA) method for the detection of EHEC (EHEC EIA) are AOAC INTERNATIONAL Official Methods 996.09 and 996.10, respectively. A minor modification to the enrichment medium used in both methods has been developed. This modification, the BioControl modified EHEC medium (BioControl mEHEC) provides a more cost-effective procedure with performance equivalent to that of the cultural method for detection of E. coli O157:H7 in beef.     

Evaluation of the VIDAS Listeria (LIS) immunoassay for the detection of Listeria in foods using demi-Fraser and Fraser enrichment broths, as modification of AOAC Official Method 999.06 (AOAC Official Method 2004.06)

A multilaboratory study was conducted to compare the VIDAS LIS immunoassay with the standard cultural methods for the detection of Listeria in foods using an enrichment modification of AOAC Official Method 999.06. The modified enrichment protocol was implemented to harmonize the VIDAS LIS assay with the VIDAS LMO2 assay. Five food types--brie cheese, vanilla ice cream, frozen green beans, frozen raw tilapia fish, and cooked roast beef--at 3 inoculation levels, were analyzed by each method. A total of 15 laboratories representing government and industry participated. In this study, 1206 test portions were tested, of which 1170 were used in the statistical analysis. There were 433 positive by the VIDAS LIS assay and 396 positive by the standard culture methods. A Chi-square analysis of each of the 5 food types, at the 3 inoculation levels tested, was performed. The resulting average Chi square analysis, 0.42, indicated that, overall, there are no statistical differences between the VIDAS LIS assay and the standard methods at the 5% level of significance.     

Equivalence of Visual Immunoprecipitate Assay (VIP) for Salmonella for the detection of motile and nonmotile Salmonella in all foods to AOAC culture method: collaborative study

Six foods representative of a wide variety of processed, dried powder processed, and raw food types were analyzed by the Visual Immunoprecipitate Assay (VIP) for Salmonella and AOAC INTERNATIONAL culture method. Paired samples of each food type were simultaneously analyzed; one sample by the VIP method and one by the AOAC culture method. A total of 24 laboratories representing federal government agencies and private industry, in the United States and Canada, participated in this collaborative study. Food types were inoculated with species of Salmonella with the exception of raw ground chicken, which was naturally contaminated. No statistical differences (p < 0.05) were observed between VIP for Salmonella interpretation and the AOAC culture method for any inoculation level of any food type or naturally contaminated food. The method was adopted Official First Action status by AOAC INTERNATIONAL.     

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.     

Comparison of visual immunoassay and chromogenic culture medium for the presence of Listeria spp. in foods

Two rapid screening methods [the TECRA Listeria Visual Immunoassay (LIS-VIS) kit, an AOAC-approved 48 h visual test, which detects Listeria through colorimetry, and BCM Listeria isolation and differentiation plating agar] were used to screen U.S. Food and Drug Administration-regulated commodities for the presence of Listeria spp. Seventy-four different food samples were screened for the presence of Listeria spp. by using both protocols. Test results for the TECRA LIS-VIA showed 66 negative samples and 1 false positive, with 4 confirmed as L. monocytogenes and 3 as L. innocua. With the BCM agar, 67 samples were negative, 4 were confirmed as L. monocytogenes, and 3 were confirmed as L. innocua. Both methods showed similar results and were effective screening tools for Listeria spp. in foods. The BCM agar method proved to be a rapid, sensitive, and excellent tool for early screening and differentiation of Listeria spp. present in foods.     

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.     

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.     

Sensitivity and specificity of the BAX for screening/Listeria monocytogenes assay: internal validation and independent laboratory study

In this study to certify the BAX for Screening/Listeria monocytogenes assay (DuPont Qualicon, Wilmington, DE), an internal evaluation was conducted on 16 food types that were simultaneously analyzed with the BAX system (BAX), and the ISO method for the detection of L. monocytogenes (ISO). No statistically significant difference in performance between the BAX and ISO methods was observed. Inclusivity/exclusivity testing showed that the BAX system was able to detect 97 of 97 (100%) of L. monocytogenes strains tested. None of 56 other Listeria species or non-Listeria tested gave a reproducible positive BAX result. Ruggedness testing demonstrated that performance of the assay was not affected by reasonable variability in the operating parameters. BAX was then submitted for independent laboratory validation. In this phase, BAX was compared with standard culture methods for the detection of L. monocytogenes in chicken (USDA-FSIS), crab meat (BAM), and milk (AOAC). This study validated product claims of sensitivity and specificity >98% in accordance with AOAC Performance Tested Method requirements.     

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.     

Effects of Ultraviolet-B Exposure on the Resistance to Listeria monocytogenes in the Rat

A rat infection model using the bacterial pathogen Listeria monocytogenes was employed to analyze the im-munosuppressive activity of UVB radiation. Rats were exposed to suberythemal doses of UVB radiation for 5 or 7 consecutive days, using Kromayer or FS40 lamps respectively. Subsequently, the rats were infected subcuta-neously or intravenously with Listeria . Exposure to UVB resulted in an increased number of bacteria in the spleen 4 days after infection. Listeria -specific lymphocyte proliferation assays as well as delayed-type hypersensitivity reactions demonstrated that T cell-mediated immunity to Listeria was impaired by UVB as measured 4 and 8 days after infection. In addition, UVB exposure decreased phagocytotic activity of peripheral blood macrophages. This study demonstrated that suberythemal doses of UVB radiation caused a delay in the clearance of Listeria bacteria from the spleen of the rats and that this was probably caused by impaired nonspecific phagocytosis of Listeria by macrophages in addition to an impaired activity of Listeria -specific T cells.