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.     

Comparative validation study to demonstrate the equivalence of a minor modification to AOAC Method 997.03 [Visual Immunoprecipitate (VIP) for Listeria] to the reference culture methods

The Visual Immunoprecipitate (VIP) for the Detection of Listeria in Foods and Environmental Surfaces, AOAC Official Method 997.03, 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 methods. Two food matrixes and one environmental surface were analyzed. In total, valid results were obtained from 145 samples and controls. Results showed that the modified VIP for Listeria spp. is equivalent to the reference culture methods for the detection of Listeria.     

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.     

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.     

Modification to the AOAC Sporicidal Activity of Disinfectants Test (Method 966.04): collaborative study

In an effort to improve AOAC Method 966.04, the Sporicidal Activity of Disinfectants Test, selected modifications to the procedure were evaluated in a collaborative study. Method 966.04 is used to generate efficacy data to support the product registration of sporicides and sterilants. The method is a carrier-based test that provides a qualitative measure of product efficacy against spores of Bacillus subtilis and Clostridium sporogenes. The use of garden soil extract and the lack of standard procedures for the enumeration of spores and neutralization of the test chemicals have been considered problematic for many years. The proposed modifications were limited to the B. subtilis and hard surface carrier (porcelain penicylinder) components of the method. The study included the evaluation of a replacement for soil extract nutrient broth and an establishment of a minimum spore titer per carrier, both considered crucial for the improvement and utilization of the method. Additionally, an alternative hard surface material and a neutralization confirmation procedure were evaluated. To determine the equivalence of the proposed alternatives to the standard method, 3 medium/carrier combinations, (1) soil extract nutrient broth/porcelain carrier (current method), (2) nutrient agar amended with 5 microg/mL manganese sulfate/porcelain carrier, and (3) nutrient agar amended with 5 microg/mL manganese sulfate/stainless steel carrier were analyzed for carrier counts, HCI resistance, efficacy, quantitative efficacy, and spore wash-off. The test chemicals used in the study represent 3 chemical classes and are commercially available antimicrobial liquid products: sodium hypochlorite (bleach), glutaraldehyde, and a combination of peracetic acid and hydrogen peroxide. Four laboratories participated in the study. The results of the spore titer per carrier, HCI resistance, efficacy, and wash-off studies demonstrate that amended nutrient agar in conjunction with the porcelain is comparable to the current method, soil extract nutrient broth/porcelain. The nutrient agar method is simple, inexpensive, reproducible, and provides an ample supply of high quality spores. Due to the current use of porcelain carriers for testing C. sporogenes, it is advisable to retain the use of porcelain carriers until stainless steel can be evaluated as a replacement carrier material for Clostridium. The evaluation of stainless steel for Clostridium has been initiated by the Study Director. Study Director recommendations for First Action revisions are provided in a modified method.     

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.     

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.     

Extension of AOAC Official Method 996.01 to the analysis of Standard Reference Material (SRM) 1846 and infant formulas.

There is currently no official method for the analysis of fatty acids (including trans fatty acids) in infant formulas. AOAC Official Method 996.01 for Fat Analysis in Cereal Products was extended to the analysis of milk-based infant formula Standard Reference Material (SRM)1846 to determine its applicability for use with infant formulas. Following the analysis of SRM 1846, 2 infant formulas, one milk-based liquid and one soy-based powdered infant formula, were analyzed for total fatty acid composition. Fatty acid methyl esters were prepared and analyzed by gas chromatography. The results of the analysis of SRM 1846 show that the mean analyzed values were highly reproducible as indicated by low coefficients of variation (CV). The CVs were <5% for the major fatty acids. Mean analyzed values for individual fatty acids in SRM 1846 were within +/- 1 standard deviation of the certificate values. The analyzed value for total fat as triglycerides (26.27 +/- 0.25%) agreed well with the certificate value (27.1 +/- 0.59%). Analyses of infant formulas showed that the concentrations of linoleic acid and fat meet the requirements for such formulas.     

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

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

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.     

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.     

Pantothenic acid (vitamin B5) in fortified foods: comparison of a novel ultra-performance liquid chromatography-tandem mass spectrometry method and a microbiological assay (AOAC Official Method 992.07)

A novel method was developed and single-laboratory validated for the determination of free pantothenic acid (vitamin B5) in a wide range of infant and adult fortified food products. The method combines simple sample preparation and chromatographic analysis using ultra-performance LC coupled to tandem MS with positive electrospray ionization. Pantothenic acid was quantified using [13C6, 15N2]-pantothenic acid as an internal standard. Calibration curves were linear between 0.08 and 1.2 microg/mL (r2 = 0.9998), and average recovery varied between 95 and 106%. The method exhibited overall RSD(r) of 1.1% and RSD intermediate reproducibility from 2.5 to 6.0% in infant formulas and cereals. Comparison of results between total and free pantothenic acid showed that the analysis of free pantothenic acid gave a good estimation of total pantothenic acid in the range of products analyzed. The method provides reliable free pantothenic acid results in a wide range of fortified foods (infant and adult nutritionals, cereal products and beverages), and shows good correlation with the microbiological method AOAC Official Method 992.07. It is a more selective, faster, and robust alternative to microbiological determination.     

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.     

Molar absorptivities of aflatoxins B1, B2, G1, and G2 in acetonitrile, methanol, and toluene-acetonitrile (9 + 1) (modification of AOAC Official Method 971.22): collaborative study

Four laboratories participated in a mini-collaborative study of AOAC Official Method 971.22, Standards for Aflatoxins, Thin-Layer Chromatographic Method, to extend the method to 3 replacement solvents for benzene for calibration of standard aflatoxin solutions. Triplicate test sample vials, each containing 25 micrograms of the respective aflatoxin for each of the 4 aflatoxins and for each of the solvents, were prepared and sent to each collaborator. The collaborators dissolved the aflatoxin in each vial in 2 mL solvent, measured the UV spectrum, and reported the absorptivity maxima near 350 nm. The concentrations of the aflatoxins in the test samples were determined by dissolving identical test samples in benzene-acetonitrile (98 + 2) and following the procedure described in AOAC Official Method 971.22. These concentrations were, in turn, used to determine the molar absorptivities in the other 3 solvents (see Table 1). AOAC Official Method 971.22 has been modified to extend its applicability to 3 replacement solvents for benzene for calibration of standard aflatoxin solutions.     

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.     

Method modification (2004.08) to field testing of visible powders on a variety of nonporous environmental surfaces: field study

The RAMP Anthrax Test Cartridge for detecting Bacillus anthracis was validated for use in the field for detection of B. anthracis spores in visible powder residues on 7 nonporous environmental surfaces. Six teams of trained first responders and civil support personnel in Class C personal protective equipment sampled visible powder residues on plastic, stainless steel, ceramic tile, wood, rubber, sealed concrete, and food-grade painted wood and analyzed the samples on the RAMP Anthrax Test System. The accuracy for each surface was at least 97% and the overall average was 98.8%. The overall average false-positive rate was 1.79% and false-negative rate was 1.07% for all surfaces. There were no significant differences between surfaces or between spore levels.     

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

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

Evaluation of VIDAs Immuno-concentration Salmonella assay Plus selective plate method (Hektoen enteric,bismuth sulfite,Salmonella identification) for detection of Salmonella in selected foods: collaborative study

The VIDAS Immuno-concentration Salmonella (ICS) plus selective plate method (Hektoen enteric, bismuth sulfite, Salmonella identification) method for the detection of Salmonella was compared to the Bacteriological Analytical Manual (BAM)/AOAC culture method in a collaborative study. Thirty-two laboratories participated in the evaluation. Each laboratory tested one or more of the 6 test products: milk chocolate, nonfat dry milk, dried whole egg, soy flour, ground black pepper, and ground raw turkey. The 2 methods were in agreement for 1,283 of the 1,440 test samples. Of the 157 test samples not in agreement, 82 were VIDAS ICS plus selective plate-positive and BAM/AOAC-negative, and 75 were VIDAS ICS plus selective plate-negative and BAM/AOAC-positive.     

Evaluation of VIDAS Immuno-Concentration Salmonella (ICS) plus selective plate method (Hektoen enteric, bismuth sulfite, Salmonella identification) for detection of Salmonella in selected foods (Method Modification 2001.07): collaborative study

A new method for detection of Salmonella in foods in 48 h has been granted AOAC First Action approval in selected foods (Official Method 2001.07) using both the VIDAS Immuno-Concentration Salmonella (ICS) method and a combination of 3 selective plates: Hektoen enteric (HE), bismuth sulfite (BS), and Salmonella Identification (SMID).