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.

Validation of a modification to Performance-Tested Method 070601: Reveal Listeria Test for detection of Listeria spp. in selected foods and selected environmental samples.

A modification to Performance-Tested Method (PTM) 070601, Reveal Listeria Test (Reveal), is described. The modified method uses a new media formulation, LESS enrichment broth, in single-step enrichment protocols for both foods and environmental sponge and swab samples. Food samples are enriched for 27-30 h at 30 degrees C and environmental samples for 24-48 h at 30 degrees C. Implementation of these abbreviated enrichment procedures allows test results to be obtained on a next-day basis. In testing of 14 food types in internal comparative studies with inoculated samples, there was a statistically significant difference in performance between the Reveal and reference culture [U.S. Food and Drug Administration's Bacteriological Analytical Manual (FDA/BAM) or U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS)] methods for only a single food in one trial (pasteurized crab meat) at the 27 h enrichment time point, with more positive results obtained with the FDA/BAM reference method. No foods showed statistically significant differences in method performance at the 30 h time point. Independent laboratory testing of 3 foods again produced a statistically significant difference in results for crab meat at the 27 h time point; otherwise results of the Reveal and reference methods were statistically equivalent. Overall, considering both internal and independent laboratory trials, sensitivity of the Reveal method relative to the reference culture procedures in testing of foods was 85.9% at 27 h and 97.1% at 30 h. Results from 5 environmental surfaces inoculated with various strains of Listeria spp. showed that the Reveal method was more productive than the reference USDA-FSIS culture procedure for 3 surfaces (stainless steel, plastic, and cast iron), whereas results were statistically equivalent to the reference method for the other 2 surfaces (ceramic tile and sealed concrete). An independent laboratory trial with ceramic tile inoculated with L. monocytogenes confirmed the effectiveness of the Reveal method at the 24 h time point. Overall, sensitivity of the Reveal method at 24 h relative to that of the USDA-FSIS method was 153%. The Reveal method exhibited extremely high specificity, with only a single false-positive result in all trials combined for overall specificity of 99.5%.

Validation of a modification to Performance-Tested Method 010403: microwell DNA hybridization assay for detection of Listeria spp. in selected foods and selected environmental surfaces.

A modification to Performance-Tested Method 010403, GeneQuence Listeria Test (DNAH method), is described. The modified method uses a new media formulation, LESS enrichment broth, in single-step enrichment protocols for both foods and environmental sponge and swab samples. Food samples are enriched for 27-30 h at 30 degrees C, and environmental samples for 24-48 h at 30 degrees C. Implementation of these abbreviated enrichment procedures allows test results to be obtained on a next-day basis. In testing of 14 food types in internal comparative studies with inoculated samples, there were statistically significant differences in method performance between the DNAH method and reference culture procedures for only 2 foods (pasteurized crab meat and lettuce) at the 27 h enrichment time point and for only a single food (pasteurized crab meat) in one trial at the 30 h enrichment time point. Independent laboratory testing with 3 foods showed statistical equivalence between the methods for all foods, and results support the findings of the internal trials. Overall, considering both internal and independent laboratory trials, sensitivity of the DNAH method relative to the reference culture procedures was 90.5%. Results of testing 5 environmental surfaces inoculated with various strains of Listeria spp. showed that the DNAH method was more productive than the reference U.S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) culture procedure for 3 surfaces (stainless steel, plastic, and cast iron), whereas results were statistically equivalent to the reference method for the other 2 surfaces (ceramic tile and sealed concrete). An independent laboratory trial with ceramic tile inoculated with L. monocytogenes confirmed the effectiveness of the DNAH method at the 24 h time point. Overall, sensitivity of the DNAH method at 24 h relative to that of the USDA-FSIS method was 152%. The DNAH method exhibited extremely high specificity, with only 1% false-positive reactions overall.