Modification of the BAX System PCR assay for detecting Salmonella in beef, produce, and soy protein isolate. Performance Tested Method 100201

The BAX System PCR assay for Salmonella detection in foods was previously validated as AOAC Research Institute (RI) Performance Tested Method (PTM) 100201. New studies were conducted on beef and produce using the same media and protocol currently approved for the BAX System PCR assay for E. coli O157:H7 multiplex (MP). Additionally, soy protein isolate was tested for matrix extension using the U.S. Food and Drug Administration-Bacteriological Analytical Manual (FDA-BAM) enrichment protocols. The studies compared the BAX System method to the U.S. Department of Agriculture culture method for detecting Salmonella in beef and the FDA-BAM culture method for detecting Salmonella in produce and soy protein isolate. Method comparison studies on low-level inoculates showed that the BAX System assay for Salmonella performed as well as or better than the reference method for detecting Salmonella in beef and produce in 8-24 h enrichment when the BAX System E. coli O157:H7 MP media was used, and soy protein isolate in 20 h enrichment with lactose broth followed by 3 h regrowth in brain heart infusion broth. An inclusivity panel of 104 Salmonella strains with diverse serotypes was tested by the BAX System using the proprietary BAX System media and returned all positive results. Ruggedness factors involved in the enrichment phase were also evaluated by testing outside the specified parameters, and none of the factors examined affected the performance of the assay.     

DuPont Qualicon BAX System assay for genus Listeria 24E

The new BAX System PCR Assay for Genus Listeria 24E was evaluated for detecting Listeria spp. in frankfurters, spinach, cooked shrimp, queso fresco cheese, and on stainless steel surfaces with a single-stage enrichment in BAX System 24 Listeria Enrichment Broth (24 LEB). Method comparison studies performed on samples with low-level inoculates showed that the BAX System demonstrates a sensitivity equivalent or superior to the U.S. Food and Drug Administration's Bacteriological Analytical Manual and the U.S. Department of Agriculture-Food Safety and Inspection Service culture methods, but with a significantly shorter time to result. Tests to evaluate inclusivity and exclusivity returned no false-negative and no false-positive results on a diverse panel of isolates, and tests for lot-to-lot variability and tablet stability demonstrated consistent performance. Ruggedness studies determined that none of the factors examined, within the range of deviations from specified parameters examined, affect the performance of the assay.     

Modification of the BAX Salmonella test kit to include a hot start functionality (modification of AOAC Official Method 2003.09)

In 2010, the BAX System PCR assay for Salmonella was modified to include a hot start functionality designed to keep the reaction enzyme inactive until PCR begins. To validate the assay's Official Methods of Analysis status to include this procedure modification, an evaluation was conducted on four food types that were simultaneously analyzed with the BAX System and either the U.S. Food and Drug Administration's Bacteriological Analytical Manual or the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook reference method for detecting Salmonella. Identical performance between the BAX System method and the reference methods was observed. Additionally, lysates were analyzed using both the BAX System Classic and BAX System Q7 instruments with identical results using both platforms for all samples tested. Of the 100 samples analyzed, 34 samples were positive for both the BAX System and reference methods, and 66 samples were negative by both the BAX System and reference methods, demonstrating 100% correlation. No instrument platform variation was observed. Additional inclusivity and exclusivity testing using the modified test kit demonstrated the test kit to be 100% accurate in evaluation of test panels of 352 Salmonella strains and 46 non-Salmonella strains.     

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.     

Sensitivity and specificity of the test kit BAX for screening/E. coli O157:H7 in ground beef: independent laboratory study

An independent laboratory study of the BAX for Screening/E. coli O157:H7 kit was conducted at the National Food Laboratory, Inc., Dublin, CA, to complete AOAC Performance Tested Method certification. The BAX system kit was compared with the BAM culture method and a modified BAM culture method for detection of E. coli O157:H7 in ground beef. The BAX system kit detected the target organism at levels approximately 10-fold lower than those that gave positive BAM results. This study validated product claims, and Performance Tested Method status was granted.     

Evaluation of the BAX system for detection of Salmonella in selected foods: collaborative study

A multilaboratory study was conducted to compare the automated BAX System to the standard cultural methods for detection of Salmonella in selected foods. Five food types--frankfurters, raw ground beef, mozzarella cheese, raw frozen tilapia fish, and orange juice--at 3 inoculation levels, were analyzed by each method. A sixth food type, raw ground chicken, was tested using 3 naturally contaminated lots. A total of 16 laboratories representing government and industry participated. In this study, 1386 samples were analyzed, of which 1188 were paired samples and 198 were unpaired samples. Of the 1188 paired samples, 461 were positive by both methods and 404 were negative by both methods. Thirty-seven samples were positive by the BAX System but negative by the standard reference method, and 11 samples were positive by standard cultural method and negative by the BAX System. Of the 198 unpaired samples, 106 were positive by the BAX System and 60 were positive by the standard cultural method. A Chi square analysis of each of the 6 food types, at the 3 inoculation levels tested, was performed. For all foods, the BAX System demonstrated results comparable to those of the standard reference methods based on the Chi square results.     

Reveal E. coli 2.0 method for detection of Escherichia coli O157:H7 in raw beef

Reveal E. coli 2.0 is a new lateral-flow immunodiagnostic test for detection of E. coli O157:H7 and O157:NM in raw beef trim and ground beef. Compared with the original Reveal E. coli O157:H7 assay, the new test utilizes a unique antibody combination resulting in improved test specificity. The device architecture and test procedure have also been modified, and a single enrichment protocol was developed which allows the test to be performed at any point during an enrichment period of 12 to 20 h. Results of inclusivity and exclusivity testing showed that the test is specific for E. coli serotypes O157:H7 and O157:NM, with the exception of two strains of O157:H38 and one strain of O157:H43 which produced positive reactions. In internal and independent laboratory trials comparing the Reveal 2.0 method to the U.S. Department of Agriculture-Food Safety and Inspection Service reference culture procedure for detection of E. coli O157:H7 in 65 and 375 g raw beef trim and ground beef samples, there were no statistically significant differences in method performance with the exception of a single internal trial with 375 g ground beef samples in which the Reveal method produced significantly more positive results. There were no unconfirmed positive results by the Reveal assay, for specificity of 100%. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device incubation time or temperature. However, addition of the promoter reagent to the test sample prior to introducing the test device is essential to proper test performance.     

Improvement of shelf stability and processing properties of meat products by gamma irradiation

To evaluate the effects of gamma irradiation on the processing properties of meat products, emulsion-type sausage, beef patties and pork loin ham were manufactured. Most contaminated bacteria were killed by 3 kGy-irradiation to raw ground beef, and sausage can be manufactured with desirable flavor, a reduction of NaCl and phosphate, and extension of shelf life using gamma irradiation on the raw meat. The beef patties were manufactured with the addition of antioxidants (200 ppm), BHA, ascorbyl palmitate, -tocopherol, or β-carotene, and gamma-irradiation. Retardation of lipid oxidation appeared at the patties with an antioxidant. A dose of 5 kGy was observed to be as effective as the use of 200 ppm NaNO₂ to provide and maintain the desired color of the product during storage. After curing, irradiation, heating and smoking could extensively prolong the shelf life of the hams.     

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.     

Comparison of assurance gold salmonella EIA, BAX for screening/Salmonella, and GENE-TRAK Salmonella DLP rapid assays for detection of Salmonella in alfalfa sprouts and sprout irrigation water

The Assurance Gold Salmonella EIA, BAX for Screening/Salmonella, and GENE-TRAK Salmonella DLP rapid assays were compared with official cultural methods described in the Bacteriological Analytical Manual (BAM) for analysis of alfalfa sprouts and sprout irrigation water for the presence of Salmonella. The lower limits of detection of 4 serovars of Salmonella cells (S. tennessee, S. muenchen, S. mbandanka, and S. cubana) in pure culture were determined as approximately log10 2, 5, and 6 for the BAX, GENE-TRAK, and Gold EIA, respectively. Despite its low detection limit, the BAX did not perform as well as the other assays in analyzing contaminated sprouts and sprout irrigation water. For 4 different lots of sprouts and sprout irrigation water samples inoculated with the 4 serovars at low [1-2 colony forming units (CFU/g)] and high (68-180 CFU/g) levels, the BAX detected Salmonella in 58/64 (90.6%) of the samples, compared with 64/64 (100%) by the GENE-TRAK, Gold EIA, and BAM methods. Assay performance was also compared for analysis of naturally contaminated sprouts and sprout irrigation water with 3 lots of alfalfa sprouted seeds associated with different salmonellosis outbreaks. Positive assay results for the naturally contaminated samples were Gold EIA 41, GENE-TRAK 36, BAM 33, and BAX 13.     

A comparison of the FAST,Premi® and KIS™ tests for screening antibiotic residues in beef kidney juice and serum

Three microbial inhibition-based screening methods, the fast antimicrobial screening test (FAST), the Premi test, and the kidney inhibition swab (KIS) test, were evaluated using penicillin G, sulfadimethoxine, oxytetracyline, tylosin, danofloxacin, streptomycin, neomycin, and spectinomycin at a range of fortified concentrations in beef kidney juice and beef serum. Each antibiotic was individually tested simultaneously using the different assays in replicate experiments. Detection threshold concentrations for each analyte in each screening assay were determined for the different matrices. Each assay gave a different detectability profile for the different antibiotics, with the largest differences related to neomycin, which was more sensitively detected by the FAST, and penicillin G, which was detected at lower levels by the Premi and KIS tests. In addition to practical considerations, analysts can use the information presented in this study to evaluate each kit for applicability to their monitoring needs.     

Sensitivity and specificity of the Sanita-kun Aerobic Count: internal validation and independent laboratory study

The Sanita-kun Aerobic Count consists of a transparent cover film, an adhesive sheet, a layer of nonwoven fabric, and a water-soluble compound film, including a culture medium formula for detection of aerobic microorganisms. The Sanita-kun sheet was validated for 14 food categories in an internal study and an independent study was conducted on ground beef and hot dogs. Both studies showed no significant difference in performance between 5 or 8 replicates of the Sanita-kun sheets and AOAC Method 966.23, excluding some lots of foods. The correlation coefficient to plate count agar in the internal accuracy study was 0.99. The average relative standard deviation for repeatability of total foods was 0.26 and 0.19, respectively, excluding < 10 average counts. The ruggedness study, which examined the influence of incubation temperature and period, recommended incubation of the Sanita-kun sheet at 32.5 +/- 2.5 degrees C for 46 +/- 2 h. Comparison of 3 lots of Sanita-kun sheets showed no decrease of performance in the older lot. The shelf-life of the sheet is at least 14 months. The Sanita-kun Aerobic Counts has been granted AOAC Performance Tested Method status.     

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.     

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.     

Evaluation of the GeneQuence DNA hybridization method in conjunction with 24-hour enrichment protocols for detection of Salmonella spp. in select foods: collaborative study

A multilaboratory study was conducted to compare performance of the GeneQuence DNA hybridization (DNAH) method incorporating new 24 h enrichment protocols and reference culture procedures for detection of Salmonella spp. in select foods. Six food types (raw ground turkey, raw ground beef, dried whole egg, milk chocolate, walnuts, and dry pet food) were tested by the DNAH method and by the culture methods of either the U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) or the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA/BAM). Fifteen laboratories participated in the study. Four of the foods tested (raw ground turkey, dried whole egg, milk chocolate, and dry pet food), showed no statistically significant differences in performance between the DNAH method and the reference procedure as determined by Chi square analysis. Sensitivity rates for the DNAH method ranged from 92 to 100%. The DNAH method, with the specific enrichment protocol evaluated, was found to be ineffective for detection of Salmonella spp. in walnuts. For raw ground beef, results from one trial showed a statistically significant difference in performance, with more positives obtained by the reference method. However, evidence suggests that the difference in the number of positives was likely due to lack of homogeneity of the test samples rather than to DNAH method performance.     

Reveal Salmonella 2.0 test for detection of Salmonella spp. in foods and environmental samples. Performance Tested Method 960801

Reveal Salmonella 2.0 is an improved version of the original Reveal Salmonella lateral flow immunoassay and is applicable to the detection of Salmonella enterica serogroups A-E in a variety of food and environmental samples. A Performance Tested Method validation study was conducted to compare performance of the Reveal 2.0 method with that of the U.S. Department of Agriculture-Food Safety and Inspection Service or U.S. Food and Drug Administration/Bacteriological Analytical Manual reference culture methods for detection of Salmonella spp. in chicken carcass rinse, raw ground turkey, raw ground beef, hot dogs, raw shrimp, a ready-to-eat meal product, dry pet food, ice cream, spinach, cantaloupe, peanut butter, stainless steel surface, and sprout irrigation water. In a total of 17 trials performed internally and four trials performed in an independent laboratory, there were no statistically significant differences in performance of the Reveal 2.0 and reference culture procedures as determined by Chi-square analysis, with the exception of one trial with stainless steel surface and one trial with sprout irrigation water where there were significantly more positive results by the Reveal 2.0 method. Considering all data generated in testing food samples using enrichment procedures specifically designed for the Reveal method, overall sensitivity of the Reveal method relative to the reference culture methods was 99%. In testing environmental samples, sensitivity of the Reveal method relative to the reference culture method was 164%. For select foods, use of the Reveal test in conjunction with reference method enrichment resulted in overall sensitivity of 92%. There were no unconfirmed positive results on uninoculated control samples in any trials for specificity of 100%. In inclusivity testing, 102 different Salmonella serovars belonging to serogroups A-E were tested and 99 were consistently positive in the Reveal test. In exclusivity testing of 33 strains of non-salmonellae representing 14 genera, 32 were negative when tested with Reveal following nonselective enrichment, and the remaining strain was found to be substantially inhibited by the enrichment media used with the Reveal method. Results of ruggedness testing showed that the Reveal test produces accurate results even with substantial deviation in sample volume or device development time.     

Determination of rendering plant sterilization conditions using a commercially available ELISA test kit developed for detection of cooked beef.

A commercially available enzyme-linked immunosorbent assay (ELISA) test kit developed for detection of cooked beef in meat samples was used to determine appropriate heat treatment of rendered materials. An improved extraction procedure increased the absolute difference in R-values between 2 rendered materials treated under different conditions (average temperature 129 and 134 degrees C, respectively). To evaluate the influence of the main sterilization parameters on ELISA results, a factorial design approach was used. The parameters investigated were temperature, time, particle size, and meat composition. Lean meat samples containing beef and pork were sterilized under strictly controlled conditions in a laboratory autoclave. The experiments demonstrated that the R-values obtained with the ELISA test kit for beef are strongly influenced by temperature and time, whereas particle size has a minor influence. The proportion of bovine material did not have any impact on R-values. Autoclave-processed lean meat samples were analyzed by using an ELISA test kit for pork, which was validated in a collaborative trial. The ELISA test kit for pork proved to be more sensitive for the investigated parameters, thus verifying and extending previous investigations.     

GHD室温自交联乳液的聚合及贮存稳定性

采用半连续种子乳液聚合技术合成了含甲基丙烯酸缩水甘油酯(GMA)、甲基丙烯酸羟乙酯(HEMA)和甲基丙烯酸二甲氨基乙酯(DMAEMA)的室温自交联乳液(GHD).实验结果表明,在甲基丙烯酸甲酯(MMA)-丙烯酸丁酯(BA)-GMA种子乳液存在下,聚合温度升高,聚合过程稳定性下降,但乳液的贮存稳定性提高;乳化单体滴加速度加快,种子聚合物的玻璃化温度升高,可减少聚合过程的交联凝聚作用,提高聚合过程的稳定性;而HEMA和DMAEMA用量增加对聚合过程的稳定性没有明显影响,但使乳液的贮存稳定性下降.官能团间的交联凝聚作用可能是影响室温自交联乳液聚合及贮存过程稳定性的关键因素.     

Design,synthesis, cytotoxic activity,and apoptosis inducing effects of 4- and N-substituted benzoyltaurinamide derivatives

In this study, a group of 4-substituted benzoyltaurinamide derivatives were designed, synthesized, and investigated for their anticancer activity against three cancer cell lines and one nontumorigenic cell line by MTT assay. Among the final compounds, methoxyphenyl derivatives 14, 15, 16 were found to be effective against all the tested cancerous cell lines with promising selectivity. The most active compounds were further evaluated to determine the molecular mechanism of their anticancer activity by using western blot assay and the Annexin V-FITC/PI test. Compound 14 (in SH-SY5Y and MDA-MB-231 cell lines) and 15 (in SH-SY5Y cell line) were found to induce intrinsic apoptotic pathway by upregulating BAX, caspase-3, and caspase-9, while downregulating Bcl-2 and Bcl-xL expression levels. According to mechanistic studies, compounds displayed their anticancer activity via three different mechanisms: a. caspase-dependent, b. caspase-independent, and c. caspase-dependent pathway that excluded caspase-9 activation. As a result, this study provides interesting data which can be used to design new taurine-based anticancer derivatives.     

Immunoassay kit used to detect the presence of bovine material in processed foods

The Tepnel Bio Kit for the detection of beef in cooked foods was assessed to determine its validity in demonstrating if food being imported into New Zealand contains beef material. The test suffered no interference from the presence of other common nonbovine species meats accepted as food within New Zealand and it detected beef in cooked samples of mixed meats when the proportion of beef in the mixture was >2 or >1%, depending on other meat species present. The documentation supplied with the kit indicates that the specific proteins it measures in cooked beef are stable to 130 degrees C. This was confirmed in the literature when the kit was used to test meat and bone meal cooked to at least 133 degrees C. However, our results showed these proteins to be much less stable when heated to elevated temperatures in moist food under pressure, and samples containing beef ceased to be positive by the immunoassay test after being autoclaved to 121 degrees C. This suggests that the test may not be able to detect even relatively high levels of beef in low-acid canned foods, which are normally retorted under pressure to approximately 121 degrees C.