Detection of Salmonella species in a variety of foods by the DuPont Bax system real-time PCR assay for Salmonella: first action 2013.02.

A multilaboratory study was conducted to evaluate the ability of the DuPont BAX System Real-Time PCR Assay for Salmonella to detect the target species in a variety of foods and environmental surfaces. Internal validation studies were performed by DuPont Nutrition & Health on 24 different sample types to demonstrate the reliability of the test method among a wide variety of sample types. Two of these matrixes-pork and turkey frankfurters and pasteurized, not-from-concentrate orange juice without pulp-were each evaluated in 14 independent laboratories as part of the collaborative study to demonstrate repeatability and reproducibility of the internal laboratory results independent of the end user. Frankfurter samples were evaluated against the U.S. Department of Agriculture, Food Safety and Inspection Service reference method as a paired study, while orange juice samples were evaluated against the U.S. Food and Drug Administration reference method as an unpaired study, using a proprietary media for the test method. Samples tested in this study were artificially inoculated with a Salmonella strain at levels expected to produce low (0.2-2.0 CFU/test portion) or high (5 CFU/test portion) spike levels on the day of analysis. For each matrix, the collaborative study failed to show a statistically significant difference between the candidate method and the reference method using the probability of detection statistical model.

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.

DuPont qualicon BAX system real-time PCR assay for Escherichia coli O157:H7.

Evaluations were conducted to test the performance of the BAX System Real-Time PCR assay, which was certified as Performance Tested Method 031002 for screening E. coli O157:H7 in ground beef, beef trim, spinach, and lettuce. Method comparison studies performed on samples with low-level inoculates showed that the BAX System demonstrates a sensitivity equivalent or superior to the FDA-BAM and the USDA-FSIS 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 affect the performance of the assay. An accelerated shelf life study determined an initial 36 month shelf life for the test kit.

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.

Evaluation of DuPont Qualicon Bax System PCR assay for yeast and mold.

Evaluations were conducted to test the performance of the BAX System PCR assay which was certified as Performance Tested Method 010902 for screening yeast and mold in yogurt, corn starch, and milk-based powdered infant formula. Method comparison studies performed on samples with low-level inoculates showed that the BAX System demonstrates a sensitivity equivalent to the U.S. Food and Drug Administration's Bacteriological Analytical Manual culture method, but with a significantly shorter time to obtain results. 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 affected the performance of the assay.

In-house validation study of the DuPont Qualicon BAX system Q7 instrument with the BAX system PCR Assay for Salmonella (modification of AOAC Official Method 2003.09 and AOAC Research Institute Performance-Tested Method 100201).

In 2006, DuPont Qualicon introduced the BAX system Q7 instrument for use with its assays. To demonstrate the equivalence of the new and old instruments, a validation study was conducted using the BAX system PCR Assay for Salmonella, AOAC Official Method 2003.09, on three food types. The foods were simultaneously analyzed with the BAX system Q7 instrument and either the U.S. Food and Drug Administration Bacteriological Analytical Manual or the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook reference method for detecting Salmonella. Comparable performance between the BAX system and the reference methods was observed. Of the 75 paired samples analyzed, 39 samples were positive by both the BAX system and reference methods, and 36 samples were negative by both the BAX system and reference methods, demonstrating 100% correlation. Inclusivity and exclusivity for the BAX system Q7 instrument were also established by testing 50 Salmonella strains and 20 non-Salmonella isolates. All Salmonella strains returned positive results, and all non-Salmonella isolates returned a negative response.

DuPont Qualicon BAX System polymerase chain reaction assay. Performance Tested Method 100201.

A recent outbreak of Salmonella in peanut butter has highlighted the need for validation of rapid detection methods. A multilaboratory study for detecting Salmonella in peanut butter was conducted as part of the AOAC Research Institute Emergency Response Validation program for methods that detect outbreak threats to food safety. Three sites tested spiked samples from the same master mix according to the U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA-BAM) method and the BAX System method. Salmonella Typhimurium (ATCC 14028) was grown in brain heart infusion for 24 h at 37 degrees C, then diluted to appropriate levels for sample inoculation. Master samples of peanut butter were spiked at high and low target levels, mixed, and allowed to equilibrate at room temperature for 2 weeks. Spike levels were low [1.08 most probable number (MPN)/25 g]; high (11.5 MPN/25 g) and unspiked to serve as negative controls. Each master sample was divided into 25 g portions and coded to blind the samples. Twenty portions of each spiked master sample and five portions of the unspiked sample were tested at each site. At each testing site, samples were blended in 25 g portions with 225 mL prewarmed lactose broth until thoroughly homogenized, then allowed to remain at room temperature for 55-65 min. Samples were adjusted to a pH of 6.8 +/- 0.2, if necessary, and incubated for 22-26 h at 35 degrees C. Across the three reporting laboratories, the BAX System detected Salmonella in 10/60 low-spike samples and 58/60 high-spike samples. The reference FDA-BAM method yielded positive results for 11/60 low-spike and 58/60 high-spike samples. Neither method demonstrated positive results for any of the 15 unspiked samples.