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.

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%.

Salmonella in foods--a new enrichment procedure for use with the TECRA Salmonella visual immunoassay: collaborative study.

A collaborative study was conducted to compare a new enrichment procedure for the TECRA Salmonella Visual Immunoassay with the reference method given in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM 7th Ed.). Three food types (milk powder, black 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. No significant differences (p > 0.05) were observed for the pairwise comparison of the proportion of positive samples for the TECRA method with that for the reference method. The new enrichment procedure for the TECRA method has been adopted First Action by AOAC INTERNATIONAL.

Dry rehydratable film method for enumerating confirmed Escherichia coli in poultry, meats, and seafood: collaborative study.

A rehydratable dry-film plating method for Escherichia coli, the Petrifilm E. coli/Coliform (EC) Count Plate in foods, has been compared with the AOAC INTERNATIONAL most probable number (MPN) method. Eleven laboratories participated in the collaborative study. Three E. coli levels in 8 samples each of frozen raw ground turkey, frozen raw ground beef, and frozen cooked fish were tested in duplicate. Mean log counts for the Petrifilm plate procedure were not significantly different from those for the MPN procedure for cooked fish samples inoculated with low or high inocula levels, for samples of raw turkey inoculated at medium level, and for beef inoculated at low, medium, and high levels. Repeatability and reproducibility variances of the Petrifilm EC Plate method recorded at 24 h were as good as or better than those of the MPN method. The dry rehydratable film method for enumerating confirmed E. coli in poultry, meats, and seafood has been adopted first action by AOAC INTERNATIONAL.

Comparison of the SimPlate total plate count method with Petrifilm, Redigel, conventional pour-plate methods for enumerating aerobic microorganisms in foods.

The SimPlate Total Plate Count (TPC) method, developed by IDEXX Laboratories, Inc., is designed to determine the most probable number of aerobic microorganisms in foods. The 24-h test was compared to the conventional plate count agar (PCA) method, the Petrifilm Aerobic Count plates, and the Redigel Total Count procedure for enumerating microflora in 751 food samples. Results using the SimPlate TPC method were highly correlated (r > or = 0.96) with results from other test methods. Slopes (0.96-0.97) were not significantly different from 1, and y intercepts (-0.03-0.08) were not different from O. The SimPlate has a high counting range (> 1600 most probable number per single dilution), thus requiring fewer dilutions of samples compared to other methods evaluated. Some foods, e.g., raw liver, wheat flour, and nuts, contain enzymes that gave false-positive reactions on SimPlates. Overall, however, the SimPlate TPC method is a suitable alternative to conventional PCA, Petrifilm, and Redigel methods for estimating populations of mesophilic aerobic microorganisms in a wide range of foods.

LOCATE enzyme-linked immunosorbent assay for detection of Salmonella in food: collaborative study.

A collaborative study was performed in 27 laboratories to validate the enzyme-linked immunosorbent procedure LOCATE for rapid detection of Salmonella in foods. Results were read visually and with a microtiter plate reader. The LOCATE method was compared with the Bacteriological Analytical Manual (BAM)/AOAC INTERNATIONAL culture method for detecting Salmonella in 6 foods: milk chocolate, nonfat dry milk, dried whole egg, soy flour, ground black pepper, and ground raw turkey. Two foods--dried whole egg and black pepper--required repeat rounds because insufficient data sets were produced initially (AOAC INTERNATIONAL stipulates a minimum of 15 sets per food type). Each laboratory tested one or more of the 6 foods. A total of 1 439 samples were analyzed, and no significant differences (P < 0.05) were observed between LOCATE with either visual or reader detection and BAM/AOAC INTERNATIONAL results. The LOCATE screening method with visual or reader detection is recommended for Official First Action Approval.

VIDAS enzyme-linked fluorescent immunoassay for detection of Salmonella in foods: collaborative study.

The VIDAS SLM method for detection of Salmonella was compared with the Bacteriological Analytical Manual (BAM)/AOAC culture method in a collaborative study. Twenty laboratories participated in the evaluation. Each laboratory tested one or more of 6 test products: milk chocolate, nonfat dry milk, dried whole egg, soy flour, ground black pepper, and ground raw turkey. No significant differences (P < 0.05) were observed between the 2 methods. The 2 methods were in agreement for 99% of 1544 samples analyzed. Of the 20 samples out of agreement, 8 were VIDAS SLM positive and BAM/AOAC negative, and 12 were VIDAS SLM negative and BAM/AOAC positive. The VIDAS SLM method for detection of Salmonella in foods has been adopted first action by AOAC INTERNATIONAL.

High-sensitivity dry rehydratable film method for enumeration of coliforms in dairy products: collaborative study.

A dry-film coliform count plate that is inoculated with 5 mL sample was compared with the Violet Red Bile Agar plate method in a collaborative study by 18 laboratories. Products analyzed were 2% milk, chocolate milk, cream, vanilla ice cream, cottage cheese, and cheese. Collaborators tested blind duplicate uninoculated samples and samples inoculated at low, medium, and high level. Significantly (P < 0.05) higher numbers of coliforms were recovered by the dry-film method from 2% milk samples at the 3 inoculum levels, the chocolate milk at the low- and high-inoculum levels, and the cream at the high-inoculum level. Significantly higher counts were obtained by the agar method for cottage cheese samples at the low-inoculum level. The repeatability standard deviation for the dry-film method was significantly higher for the high-inoculum level chocolate milk sample and the medium-inoculum level cottage cheese. The same statistic was significantly higher for the agar method at all 3 inoculum levels in the 2% milk and the medium-inoculum level cream. The high-sensitivity dry rehydratable film method for enumeration of coliforms in dairy products has been adopted first action by AOAC INTERNATIONAL.

Enzyme-linked immunoassay for detection of Listeria monocytogenes in dairy products, seafoods, and meats: collaborative study.

A collaborative study was conducted to evaluate Listeria-Tek, an enzyme-linked immunosorbent assay (ELISA) for detection of Listeria monocytogenes and other Listeria spp. in foods. The present ELISA method was compared to the U.S. Food and Drug Administration culture method for detection of L. monocytogenes in dairy products and seafoods and to the U.S. Department of Agriculture Food Safety and Inspection Service method for detection of L. monocytogenes in meats. Replicate samples of 6 food types (frankfurters, roast beef, Brie cheese, 2% milk, raw shrimp, and crab meat) inoculated with L. monocytogenes and uninoculated control samples were analyzed by the collaborators. L. monocytogenes was identified in 593 samples by the ELISA method and in 574 samples using culture procedures. Identical results were obtained for 506 positive samples and 419 negative samples using the ELISA and culture methods for an overall agreement rate of 85.6%. The enzyme-linked immunoassay for detection of L. monocytogenes in dairy, seafood, and meat products has been adopted first action by AOAC INTERNATIONAL.

Deoxyribonucleic acid hybridization method for the detection of Listeria in dairy products, seafoods, and meats: collaborative study.

The method is based on the hybridization of synthetic deoxyribonucleic acid probes to ribosomal ribonucleic acid sequences unique to Listeria. This method was compared to 2 culture methods: the U.S. Food and Drug Administration method for the detection of Listeria in dairy products and seafoods and the U.S. Department of Agriculture, Food Safety and Inspection Service method for Listeria in meats. Six food types with replicate samples containing various concentrations of Listeria were analyzed by the collaborating laboratories. Listeria was detected in 774 samples using the DNAH method and in 772 samples using a culture method. The DNAH and culture methods were in agreement for 668 samples containing Listeria and 306 samples without Listeria. The overall rate of agreement between methods was 82.3%. The method has been adopted first action by AOAC INTERNATIONAL.

Elevated-temperature, colorimetric, monoclonal, enzyme-linked immunosorbent assay for rapid screening of Salmonella in foods: collaborative study.

A collaborative study was performed by 30 laboratories in 3 sets of trials to validate a modified colorimetric monoclonal enzyme-linked immunosorbent assay (ELISA) method for Salmonella detection. The modifications to the current methodology included incubation of enrichments and post-enrichments at an elevated temperature, addition of novobiocin to the M-broth post-enrichment, and elimination of the centrifugation and agitation steps. Five artificially contaminated foods (nonfat dry milk, milk chocolate, dried egg, ground black pepper, and soy flour) and 1 naturally contaminated food (raw ground turkey) were analyzed. The artificially contaminated foods were inoculated with individual Salmonella serotypes at a high (10-50 cells/25 g) and low (1-5 cells/25 g) contamination level. Results from the modified ELISA method were compared to the Bacteriological Analytical Manual (BAM)/AOAC culture method. In 2 of the food products, milk chocolate and pepper, a number of laboratories isolated Salmonella from un-inoculated control samples, thus invalidating their data. As a result, there were too few laboratories remaining with valid data, and these foods were repeated. In the completed study, there were 11 false negative results obtained by the modified ELISA method, while there were 28 false negatives produced by the BAM/AOAC procedure. There were 11 ELISA positive assays which could not be confirmed by culture methods. Statistically, there were no differences between the modified, colorimetric, monoclonal ELISA and the reference culture method in all foods except raw turkey, where the ELISA method was more productive. The colorimetric monoclonal enzyme immunoassay (Salmonella-Tek) method for detecting Salmonella in all foods has been adopted first action by AOAC INTERNATIONAL.

Use of an Elevated Temperature and Novobiocin in a Modified Enzyme-linked Immunosorbant Assay for the Improved Recovery of Salmonella from Foods.

A modified colorimetric enzyme-linked immunosorbant assay (ELISA) for Salmonella detection was compared to the standard culture method of the Bacteriological Analytical Manual/Association of Official Analytical Chemists (BAM/AOAC) using 20 artificially contaminated foods (1,200 test samples). The modifications to the current methodology consisted of an elevated incubation temperature of 42°C for the tetrathionate selective broth and M-broth postenrichments, as well as addition of 10 µg/ml novobiocin to the M-broth. The microtiter plate as not agitated during assay incubation, and centrifugation steps were eliminated from the protocol. This modified ELISA method was at least as productive as the standard AOAC culture method for the food samples tested. No false-positive reactions were encountered. The false-negative incidence was 1.5% for the immunoassay and 5.3% by the AOAC cultural method. The incidence of agreement between the methods was 96.7%.

Dry rehydratable film for enumeration of total coliforms and Escherichia coli in foods: collaborative study.

Rehydratable dry-film plating methods for total coliforms and Escherichia coli in foods have been compared to the AOAC most probable number methods. Fourteen laboratories participated in the collaborative study. Three coliform and E. coli levels in 6 samples of 4 product types (flour, nuts, cheese, and beef with gravy) and in 3 samples of 2 product types (mushrooms and raw turkey) were tested in duplicate by the participants. The mean log counts for the 3 methods were comparable. In general, the repeatability and reproducibility variances of the plating methods were as good as or better than that of the MPN method. The method has been adopted official first action by AOAC.

Colorimetric and fluorometric substrate immunoassays for detection of Salmonella in all foods: comparative study.

Two modified fluorescent enzyme immunoassays for the detection of Salmonella in food have been developed. Both of the new procedures, which substitute a colorimetric substrate for the fluorescent substrate and in which results are read visually or with a photometer, are modifications of AOAC method 989.15. The visually read procedure uses the same antibody-coated wells as in method 989.15. The colorimetric end point of the assay is determined by comparing the solution color to a color chart. The assay result may also be read in a photometer, if the solution is first transferred to a transparent microtiter well. The second procedure designed to be read in a photometer substitutes clear, antibody-coated wells for those used in the fluorescent assay. The colorimetric assays employ identical monoclonal antibodies for capture and detection of Salmonella as used in the fluorescent assay. In this comparative study, the performance of each new assay was consistent with the performance of method 989.15. These methods have been adopted official first action by AOAC as alternative methods for the detection of Salmonella in foods.

Colorimetric deoxyribonucleic acid hybridization assay for rapid screening of Salmonella in foods: collaborative study.

A collaborative study was performed in 11 laboratories to validate a colorimetric DNA hybridization (DNAH) method for rapid detection of Salmonella in foods. The method was compared to the standard culture method for detection of Salmonella in nonfat dry milk, milk chocolate, soy isolate, dried whole egg, ground black pepper, and raw ground turkey. Samples inoculated with high (0.4-2 cells/g) and low (0.04-0.2 cells/g) levels of Salmonella and uninoculated control samples were included in each food group analyzed. There was no significant difference in the proportion of samples positive by DNAH and culture procedure for any of the 6 foods. The colorimetric DNA hybridization assay screening method has been adopted official first action as a rapid screening method for detection of Salmonella in all foods.

Dry rehydratable film for enumeration of total aerobic bacteria in foods: collaborative study.

A rehydratable dry-film plating procedure for aerobic plate counts has been compared to the standard agar plate method (966.23B and C, 15th ed.; 46.014-46.015, 14th ed.) in a collaborative study by 12 laboratories. Each laboratory analyzed the normal microflora of 3 samples in duplicate for 6 products. The aerobic plate counts ranged from 1.0 x 10(3) to 1.0 x 10(8) cfu/g. The products were flour, nuts, frozen raw shrimp, spice, frozen raw ground turkey, and frozen and refrigerated vegetables. Repeatability standard deviations of the 2 methods did not differ significantly for 13 of 18 test samples. For 1 shrimp and 2 turkey samples, the dry-film method had lower repeatability variances (P less than 0.05) and for 1 spice sample the agar method had lower repeatability variances (P less than 0.05). Relative standard deviations of repeatability were between 1.7 and 15.5% for the dry-film method and 1.2 and 16.0% for the agar method. Relative standard deviations of reproducibility ranged from 2.4 to 23.4% for the dry-film method and 2.3 to 18.8% for the agar method. The dry rehydratable film method has been adopted official first action for determination of the aerobic plate count.

Comparison of colorimetric monoclonal enzyme immunoassay screening methods for detection of Salmonella in foods.

A colorimetric enzyme immunoassay (EIA) method for detection of Salmonella in foods has been compared to the AOAC colorimetric monoclonal EIA screening method (986.35, 15th ed.; 46.B21-46.B29, 14th ed.). The assays use the same monoclonal antibodies and have similar reactivity toward Salmonella. However, the new assay uses antibody-coated microtiter wells instead of coated magnetic beads to capture Salmonella antigens. Compared with the bead assay, the coated-well assay format requires significantly less time to complete, and was consistently able to detect lower levels of Salmonella in mixed culture. Compared to the standard AOAC culture method for food samples, the plate assay was as productive. No false negatives were obtained by the immunoassay; the false negative rate was 1.1% by the culture method. The rate of agreement between the 2 methods was 99.1%. The official final action bead assay method for Salmonella in foods, 986.35, and the same assay for use with low-moisture foods, 987.11, have been modified official first action to use antibody-coated microtiter strip-wells.

Dry rehydratable films for enumeration of coliforms and aerobic bacteria in dairy products: collaborative study.

A collaborative study was conducted to compare proposed dry-film plating methods, using aerobic count plates and coliform count plates, to standard agar plating methods for quantifying aerobic bacteria and coliforms in dairy products. In this study, 5 food products (chocolate milk, pasteurized cheese, nonfat dry milk, evaporated milk, and vanilla ice cream), selected as representative dairy products, were analyzed by 11 collaborating laboratories. The results indicate that the dry-film plating methods are equivalent to or better than the agar plating methods. The aerobic count and coliform count dry-film plating methods have been adopted official first action.

Comparative studies of nucleic acid hybridization assay for Listeria in foods.

A nucleic acid hybridization assay has been developed for Listeria spp. in dairy foods and environmental samples. The assay is based on detection of unique Listeria 16S rRNA sequences by using a 32P-labeled synthetic DNA probe. Inclusivity and exclusivity of the probe were confirmed with 139 Listeria isolates representing all known species, and 73 non-Listeria bacterial strains. In this paper, we present results from our preliminary studies comparing the hybridization assay with conventional culture on a total of 575 specimens that represent a variety of inoculated and uninoculated foods and environmental samples. The assay, which is done in a filter manifold format after 2 days of cultural enrichment, requires a total assay time of less than 2.5 days. The false-negative rate for all sample groups tested using the GENE-TRAK hybridization assay was less than the rate for culture. Thus, the new assay allows rapid screening of the indicated product groups and provides reliable numerical results.

DNA hybridization assay for detection of Salmonella in foods: collaborative study.

A collaborative study was performed in 11 laboratories to validate a DNA hybridization (DNAH) procedure for detection of Salmonella in foods. The DNAH procedure was compared to the standard culture method for detection of Salmonella in 6 foods: ground pepper, soy flour, dry whole egg, milk chocolate, nonfat dry milk, and raw deboned turkey. With the exception of turkey which was naturally contaminated, uninoculated and inoculated samples of each food group were analyzed. Results for the DNAH method were significantly better than for the standard culture method at the 5% probability level for the detection of Salmonella in turkey. There was no significant difference between the methods for the other 5 foods. The method has been adopted official first action.