Comparison of modified immunodiffusion and Bacteriological Analytical Manual (BAM) methods for detection of Salmonella in raw flesh and highly contaminated food types.

A wide variety of naturally contaminated and inoculated raw flesh and highly contaminated food types was analyzed by a modified immunodiffusion enrichment protocol and the Bacteriological Analytical Manual (BAM) method to determine the equivalence of these methods. This modification was developed by Agriculture Canada to allow addition of a high-temperature selective enrichment step in tetrathionate brilliant green broth at 42 degrees C while maintaining a 2-day total test time. Foods representing red meat, white meat, frog, and seafoods and one type of animal meal were evaluated. A total of 320 samples was tested, resulting in false negative rates of 5.2 and 3.5%, respectively, for the modified immunodiffusion and the BAM culture methods. The overall agreement rate was 96.9%.

Polyclonal enzyme immunoassay method for detection of motile and non-motile Salmonella in foods: comparative study.

A new enzyme immunoassay (EIA) method for detection of motile and non-motile Salmonella was examined in a comparative study. This method uses a proprietary formulation of polyclonal antibodies to Salmonella and is controlled to maintain specificity. Sensitivity is enhanced with an additional antibody reaction designed to minimize false-negative reactions attributable to steric interference that can occur during conjugate binding in immunoassay procedures. Twenty food types representative of a wide variety of food products were analyzed by both the EIA method and the AOAC/Bacteriological Analytical Manual (BAM) method, 967.26. Of the 1000 samples analyzed, there was a 95.6% agreement rate between the EIA method and the AOAC/BAM method. False-negative rates for the 2 methods were comparable for all foods and all Salmonella levels except ground poultry, where the EIA method detected significantly more confirmed positive samples than did the AOAC/BAM method. Twenty-seven samples were positive by EIA but negative by the culture method, and 17 samples were negative by EIA but positive by the culture method. There were no false-positive isolates detected in the comparative study.

Substrate supporting disc method for confirmed detection of total coliforms and E. coli in all foods: collaborative study.

The ColiComplete substrate supporting disc (SSD) method for simultaneous confirmed total coliform count and Escherichia coli determination in all foods was compared with AOAC most probable number (MPN) methods, 966.23 and 966.24. Twenty-nine laboratories participated in this collaborative study in which 6 food types were analyzed. Four food types, raw ground beef, pork sausage, raw liquid milk, and nut meats, were naturally contaminated with coliform bacteria. Two foods, dry egg and fresh frozen vegetables, were seeded with coliforms. Three food types, ground beef, raw liquid milk, and pork sausage, were naturally contaminated with E. coli. Although pork sausage was naturally contaminated, the level was very low (<10/50 g); therefore, additional E. coli were inoculated into 1 lot of this food type. Three food types, nut meats, dry egg, and fresh frozen vegetables, were inoculated with E. coli. For naturally contaminated samples, duplicate determinations were made on 3 separate lots for each food type. For inoculated samples, low, medium, and high contamination levels plus uninoculated control samples were examined in duplicate. Data were analyzed separately for total coliform bacteria and for E. coli. Mean log MPN counts were determined by the SSD method and the appropriate AOAC MPN method. Results were then analyzed for repeatability, reproducibility, and mean log MPN statistical equivalence. Results were statistically equivalent for all total coliform levels in all food types except frozen vegetable and raw nut meat uninoculated control samples and 1 lot of pork sausage where the SSD method produced statistically significant greater numbers. For the E. coli determinations, results were statistically equivalent across all samples and all levels for each food type. The SSD method has been adopted first action by AOAC International for confirmed detection of total coliforms and E. coli in all foods.

Modified immunodiffusion method for detection of Salmonella in raw flesh and highly contaminated foods: collaborative study.

A total of 19 government and private industry laboratories in Canada and the United States participated in the collaborative study. Naturally contaminated ground poultry and animal meals, as well as inoculated raw shrimp, were examined for presence of Salmonella by both the modified immunodiffusion method and the Bacteriological Analytical Manual culture method, resulting in an agreement rate of 93.1%. The 2 methods are statistically equivalent for all food types at each inoculation level and for all lots of naturally contaminated foods evaluated in this study. The modification of the AOAC Official Method 989.13, immunodiffusion (1-2 TEST) method for detection of motile Salmonella in all foods, has been adopted revised first action by AOAC INTERNATIONAL.

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.

Equivalence of assurance Gold Enzyme Immunoassay for visual or instrumental 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 Assurance Gold Salmonella Enzyme Immunoassay (EIA) and AOAC INTERNATIONAL culture method. Paired samples of each food type were simultaneously analyzed; one sample by the Assurance method and one by the AOAC culture method. The results for Assurance method were read visually and instrumentally with a microplate reader. 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 Assurance Gold Salmonella EIA with either visual or instrumental interpretation and the AOAC culture method for any inoculation level of any food type or naturally contaminated food. The Assurance visual and instrumental options of reading sample reactions produced the same results for 1277 of the 1296 sample and controls analyzed.

Escherichia coli O157:H7 visual immunoprecipitate assay: a comparative validation study.

A wide variety of food products, with emphasis on raw meat products, were analyzed simultaneously by 3 methods: the Escherichia coli O157:H7 (EHEC) visual immunoprecipitate assay (VIP), the Assurance EHEC enzyme immunoassay, and a modified Bacteriological Analytical Manual (BAM) culture method. This paper reports results of a comparative study of the VIP and modified BAM methods. In this comparative study, 1050 samples and controls gave false-negative rates of 1.0 and 0%, respectively, for the VIP and the modified BAM culture methods. The overall agreement between the 2 methods was 99.4%. Cultural confirmation of presumptive positive samples was problematic because competitive flora were present in higher levels than EHEC in the enrichment broth after incubation and the target organism had nondescript characteristics on the primary selective agar, hemorrhagic coli agar, which necessitated picking of additional colonies in many instances.

Assurance Escherichia coli O157:H7: a comparative validation study.

A wide variety of food products, with emphasis on raw meat products, were analyzed simultaneously by 3 methods: the Assurance Escherichia coli O157:H7 (EHEC) enzyme immunosorbent assay (EIA), the EHEC visual immunoprecipitate assay (VIP), and a modified Bacteriological Analytical Manual (BAM) culture method. This paper reports results of a comparative study of the Assurance and modified BAM methods. In this comparative study, 1050 samples and controls gave false-negative rates of 1.0 and 0%, respectively, for the Assurance EIA and the modified BAM culture methods. The overall agreement between the 2 methods was 99.4%. Cultural confirmation of presumptive positive samples was problematic because competitive flora were present in higher levels than EHEC in the enrichment broth after incubation and the target organism had nondescript characteristics on the primary selective agar, hemorrhagic coli agar, which necessitated picking of additional colonies in many instances.

Assurance polyclonal enzyme immunoassay for detection of Listeria monocytogenes and related Listeria species in selected foods: collaborative study.

Six foods representing a variety of food products were analyzed by the Assurance Listeria polyclonal enzyme immunoassay (EIA) and by either the Bacteriological Analytical Manual or the U.S. Department of Agriculture culture method for detecting Listeria monocytogenes and related Listeria species. Samples of each food type, at each inoculation level, were analyzed simultaneously by both methods. A total of 19 laboratories representing federal government agencies and private industry in the United States and Canada participated. Food types were inoculated with Listeria species including L. monocytogenes, with the exception of 3 lots of green beans, which were naturally contaminated. During this study, 1764 samples and controls were analyzed and confirmed, of which 492 were positive and 947 were negative by both methods. There were 159 samples that were positive by culture method but negative by the EIA and 188 that were negative by culture method but positive by EIA. Twenty-two samples were negative by EIA and by culture method but confirmed positive when Assurance selective enrichment broths were subcultured to selective agar. The Assurance polyclonal EIA for detecting L. monocytogenes and related Listeria species in foods has been adopted first action by AOAC INTERNATIONAL.

Visual immunoprecipitate assay (VIP) for Listeria monocytogenes and related Listeria species detection in selected foods: collaborative study.

Six foods representing a variety of food products were analyzed by the Visual Immunoprecipitate Assay (VIP) and either the Bacteriological Analytical Manual (BAM) or the U.S. Department of Agriculture culture methods for detection of Listeria monocytogenes and related Listeria spp. Samples of each food type at each inoculation level were simultaneously analyzed by both methods. A total of 23 laboratories representing federal agencies and private industry in the United States and Canada participated in this collaborative study. Foods were inoculated with Listeria species including L. monocytogenes, with the exception of 3 lots of green beans that were naturally contaminated. During this study, 1509 samples and controls were analyzed and confirmed, of which 370 were positive and 921 were negative by both methods. One hundred and fifteen samples were positive by culture methods but negative by VIP. One hundred and thirty-two were negative by culture methods but positive by the VIP. Twenty-nine samples were negative by VIP and by culture methods but confirmed positive when VIP selective enrichment broths were subcultured to selective agars. The VIP method for detection of L. monocytogenes and related Listeria spp. in foods has been adopted first action by AOAC INTERNATIONAL.

Visual immunoprecipitate assay (VIP) for detection of enterohemorrhagic Escherichia coli (EHEC) O157:H7 in selected foods: collaborative study.

Five foods representative of a variety of food products were analyzed by the Visual Immunoprecipitate Assay (VIP) and the Bacteriological Analytical Manual (BAM) culture method for the presence of Escherichia coli O157:H7. A total of 21 laboratories representing state and federal government agencies, as well as private industry, in the United States and Canada participated. Food types were inoculated with strains of E. coli O157:H7, with the exception of one lot of poultry, which was naturally contaminated. During this study, a total of 1377 samples and controls were analyzed and confirmed, of which 508 were positive and 867 were negative by both methods. Two samples were positive by BAM and negative by VIP. Because of the study design, it was not possible for the BAM method to produce false-negative or false-positive results. The VIP assay for detection of EHEC in selected foods has been adopted first action by AOAC INTERNATIONAL.

Assurance enzyme immunoassay for detection of enterohemorrhagic Escherichia coli O157:H7 in selected foods: collaborative study.

Five foods types were analyzed by the Assurance EHEC (Escherichia coli O157:H7) enzyme immunoassay (EIA) and by the Bacteriological Analytical Manual (BAM) culture method. Each sample of each food type at each inoculation level was simultaneously analyzed by both methods. A total of 21 laboratories representing state and federal government agencies and private industry in the United States and Canada participated. Samples were inoculated with E. coli O157:H7, except for one lot of poultry that was naturally contaminated. A total of 1304 samples and controls were analyzed and confirmed, of which 473 were positive and 818 were negative by both methods. Thirteen samples were positive by BAM but negative by EIA. Because of the study design, it was not possible for the BAM method to produce false-negative or false-positive results. The Assurance method for detection of E. coli O157:H7 in selected foods has been adopted first action by AOAC INTERNATIONAL.

Comparative and multilaboratory studies of two immunodiffusion method enrichment protocols and the AOAC/Bacteriological Analytical Manual culture method for detection of Salmonella in all foods.

The single enrichment immunodiffusion (1-step), the preenrichment and selective enrichment immunodiffusion (2-step), and the AOAC/Bacteriological Analytical Manual culture methods for Salmonella were evaluated for equivalence in 2 separate studies, a comparative evaluation and a multilaboratory dilution study. In the comparative study, all 3 methods were performed on 10 food types. For 550 samples, analyses resulted in 99.3 and 99.6% agreement between the culture method and the 1-step and 2-step methods, respectively. False negative rates were 0.9 and 0.3% for 1-step and culture, and 0.0% and 0.6% for the 2-step and culture, respectively. Subsequently, 6 food types were included in a multilaboratory dilution-to-extinction study. A sequential dilution series of Salmonella in foods was analyzed by the 3 methods to determine their lower limits of detection for Salmonella. A total of 1185 samples analyzed resulted in 98.9% agreement between 1-step and culture, and 99.7% agreement between 2-step and culture. False negative rates were 1.8 and 0.1% for 1-step and culture, and 0.4 and 0.1% for 2-step and culture, respectively. During these evaluations, 1735 samples and controls representing 10 different naturally contaminated and inoculated foods were tested. The data indicate statistical equivalence of all 3 methods when analyzing all food types.