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.     

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 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.     

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.     

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.     

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.     

Evaluation of a chromogenic medium for identification and differentiation of Listeria monocytogenes in selected foods

Listeria monocytogenes continues to be a threat to food safety in the United States despite a "zero tolerance" policy. When Listeria species are identified by standard cultural methods, confirmation of L. monocytogenes takes days to complete. RAPID'L.Mono agar, developed by Bio-Rad Laboratories, is a chromogenic medium that differentiates L. monocytogenes from other species of Listeria by a simple color change reaction. Differentiation is based on the specific detection of phosphatidylinositol phospholipase C activity, resulting in a blue colony, and the inability of L. monocytogenes to metabolize xylose, resulting in the absence of a yellow halo. Detection principles of standard method agars, Oxford and PALCAM, are based on the ability of all species of Listeria to hydrolyze esculin. Thus, all species of Listeria have similar colony morphology on these agars, making differentiation of pathogenic L. monocytogenes from other nonhuman pathogens difficult. RAPID'L.Mono agar has been validated with surimi, mixed salad, brie, and processed deli turkey because of the prevalence of L. monocytogenes in these foods. Sensitivity and specificity for this medium was determined to be 99.4 and 100%, respectively. Overall method agreement of RAPID'L.Mono with standard culture methods (U.S. Department of Agriculture/Food Safety and Inspection Service; U.S. Food and Drug Administration/Bacteriological Analytical Manual; and AOAC INTERNATIONAL) was excellent, with enrichment protocols 24 h shorter than those of standard methods.     

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.     

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.     

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.     

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.     

Precollaborative study of the GeneQuence Salmonella assay using 24-hour enrichment protocols for detection of Salmonella spp. in select foods

New enrichment protocols are described for use with a DNA hybridization (DNAH) method for detection of Salmonella spp. in select foods. GeneQuence Salmonella, in its original version, utilized a 3-stage enrichment of minimum 42 h duration. New 2-stage procedures of 24-28 h duration are described for raw poultry, raw beef, pasteurized egg products, milk chocolate, and dry pet food. In the validation study described here, a total of 345 samples were tested by the abbreviated DNAH method in parallel with either the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA/BAM) or U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) reference culture procedures. Results showed an overall sensitivity for the DNAH method of 97.1% (false-negative rate 2.9%). There were no false-positive results by the DNAH method; therefore the specificity was 100%. Overall agreement between the DNAH and reference culture methods was 98.5%. There were no significant differences in performance between the DNAH and reference methods for any of the foods tested as determined by Chi-square analysis. It is recommended that the DNAH method be subjected to AOAC collaborative study.     

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.     

Evaluation of VIDAS Immuno-Concentration Salmonella (ICS)/VIDAS Salmonella (SLM) immunoassay method for detection of Salmonella in selected foods (Method Modification 2001.09): collaborative study

A new method for detection of Salmonella in foods in a minimum of 24 h was adopted as an AOAC Official First Action Method for selected foods (2001.09) using both the VIDAS Immuno-Concentration Salmonella (ICS) and VIDAS Salmonella (SLM) methods.     

Detection and isolation of Listeria monocytogenes from food samples: implications of sublethal injury

Detection of L. monocytogenes is often limited by the performance of the enrichment media used to support bacterial growth to detectable levels. Because Listeria may exist at extremely low levels in foods, sample enrichment protocols must amplify these low initial populations to detectable limits. Listeria may also exist in an injured state in food products as a result of processing treatments such as heating, freezing, exposure to acids, or exposure to sanitizing compounds. Selective agents in enrichment media normally used for recovery of Listeria may inhibit repair and detection of sublethally injured Listeria, which may go on to repair, grow, and regain pathogenicity. Simple modifications to existing regulatory protocols, such as those that use more than one enrichment broth, raise sensitivity of detection to 90%. This review shows the efficacy of repair/enrichment strategies, which increase sensitivity of detection to 97.5-98.8% compared with 65-70% by standard regulatory protocols. Ribotype analysis of isolates obtained from meat samples reveals a complex microbial ecology, with striking differences in both number and distribution of distinct genetic types of Listeria, depending upon whether samples are enriched in selective or repair/enrichment media. In studies on enrichment of dairy environmental samples in University of Vermont medium and Listeria repair broth (UVM and LRB), combining these 2 primary enrichment media into a single tube of Fraser broth for dual secondary enrichment yielded a significantly higher percentage (p < 0.05) of Listeria-positive samples than did use of either LRB or UVM alone. Refinement of conventional Listeria recovery methods should consider the importance of the enrichment step, the nutritional needs of specific genetic types, and the physiological condition of Listeria isolates in foods.     

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.     

Rapid identification of closely related muscle foods by vibrational spectroscopy and machine learning

Muscle foods are an integral part of the human diet and during the last few decades consumption of poultry products in particular has increased significantly. It is important for consumers, retailers and food regulatory bodies that these products are of a consistently high quality, authentic, and have not been subjected to adulteration by any lower-grade material either by accident or for economic gain. A variety of methods have been developed for the identification and authentication of muscle foods. However, none of these are rapid or non-invasive, all are time-consuming and difficulties have been encountered in discriminating between the commercially important avian species. Whilst previous attempts have been made to discriminate between muscle foods using infrared spectroscopy, these have had limited success, in particular regarding the closely related poultry species, chicken and turkey. Moreover, this study includes novel data since no attempts have been made to discriminate between both the species and the distinct muscle groups within these species, and this is the first application of Raman spectroscopy to the study of muscle foods. Samples of pre-packed meat and poultry were acquired and FT-IR and Raman measurements taken directly from the meat surface. Qualitative interpretation of FT-IR and Raman spectra at the species and muscle group levels were possible using discriminant function analysis. Genetic algorithms were used to elucidate meaningful interpretation of FT-IR results in (bio)chemical terms and we show that specific wavenumbers, and therefore chemical species, were discriminatory for each type (species and muscle) of poultry sample. We believe that this approach would aid food regulatory bodies in the rapid identification of meat and poultry products and shows particular potential for rapid assessment of food adulteration.     

Enumeration of total yeasts and molds in foods by the SimPlate Yeast and Mold-Color Indicator method and conventional culture methods: collaborative study

The relative effectiveness of the SimPlate Yeast and Mold-Color Indicator method (Y&M-CI) was compared to the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM) method and the proposed International Organization for Standardization (ISO) method, ISO/CD 21527, for enumerating yeasts and molds in foods. Test portions were prepared and incubated according to the conditions stated in both the BAM and ISO methods. Six food types were analyzed: frozen corn dogs, nut meats, frozen fruits, cake mix, cereal, and fresh cheese. Nut meats, frozen fruits, and fresh cheese were naturally contaminated. All other foods were artificially contaminated with either a yeast or mold. Seventeen laboratories throughout North America and Europe participated in the study. Three method comparisons were conducted. In general, there was <0.3 mean log count difference in recovery between the SimPlate method and the 2 corresponding reference methods. Moreover, mean log counts between the 2 reference methods were also very similar. The repeatability (Sr) and reproducibility (SR) standard deviations were comparable between the 3 method comparisons. These results indicate that the BAM method and the SimPlate method are equivalent for enumerating yeast and mold populations in foods. Similarly, the SimPlate method is comparable to the proposed ISO method when test portions are prepared and incubated as defined in the proposed ISO method.     

Optimized, one-step, recovery-enrichment broth for enhanced detection of Listeria monocytogenes in pasteurized milk and hot dogs

A one-step, recovery-enrichment broth, optimized Penn State University (oPSU) broth, was developed to consistently detect low levels of injured and uninjured Listeria monocytogenes cells in ready-to-eat foods. The oPSU broth contains special selective agents that inhibit growth of background flora without inhibiting recovery of injured Listeria cells. After recovery in the anaerobic section of oPSU broth, Listeria cells migrated to the surface, forming a black zone. This migration separated viable from nonviable cells and the food matrix, thereby reducing inhibitors that prevent detection by molecular methods. The high Listeria-to-background ratio in the black zone resulted in consistent detection of low levels of L. monocytogenes in pasteurized foods by both cultural and molecular methods, and greatly reduced both false-negative and false-positive results. oPSU broth does not require transfer to a secondary enrichment broth, making it less laborious and less subject to external contamination than 2-step enrichment protocols. Addition of 150mM D-serine prevented germination of Bacillus spores, but not the growth of vegetative cells. Replacement of D-serine with 12 mg/L acriflavin inhibited growth of vegetative cells of Bacillus spp. without inhibiting recovery of injured Listeria cells. oPSU broth may allow consistent detection of low levels of injured and uninjured cells of L. monocytogenes in pasteurized foods containing various background microflora.