Immunomagnetic separation and visual fluorescence detection of Salmonella spp., using AOAC approved SAS Molecular Tests.

The SAS Molecular Tests method for detection of Salmonella spp. in various food matrixes has been certified by the AOAC Research Institute and designated Performance Tested Method No. 021202. The current method modification includes the optional immunomagnetic separation (IMS) to enrich the bacteria as well as optional visual fluorescence readout without the use of a turbidimeter. The modifications were validated against the U.S. Department of Agriculture/ Food Safety and Inspection Service Microbiology Laboratory Guidebook (MLG) and the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) reference methods. Food matrixes (chicken carcass rinse, beef trim, and spinach) were inoculated with low levels of Salmonella spp. (0.2-2 CFU/test portion) to generate fractional positives (5-15) in 20 inoculated samples. Samples were enriched with SAS Enrichment medium and incubated at 42 +/- 1 degree C. Enrichments were tested directly and subjected to anti-Salmonella IMS prior to the SAS Molecular Tests. Results were determined via visual fluorescence and via turbidity using a turbidimeter. All replicates were confirmed using the MLG or BAM reference method procedures, regardless of presumptive result. The SAS Molecular Tests Salmonella Detection modified methods were determined to be equivalent to the reference methods for the detection of Salmonella in chicken carcass rinse, beef trim, and fresh spinach. The inclusion of IMS in the modified method improved the detection rate of Salmonella in chicken carcass rinses and spinach. The optional use of visual fluorescent reagent and heat block either with IMS or without IMS produced results that were comparable to the results obtained from using a real-time turbidimeter.

Immunomagnetic separation and visual fluorescence detection of E. coli O157 using AOAC approved SAS Molecular Tests.

The SAS Molecular Tests method for the detection of E. coli O157 in various food matrixes has been certified by the AOAC Research Institute and designated Performance Tested Method No. 031203. The current method modification includes the optional immunomagnetic separation (IMS) to enrich the bacteria as well as optional visual fluorescence readout without the use of a turbidimeter. The following study was conducted to validate the proposed modifications against the U.S. Department of Agriculture/Food Safety and Inspection Service Microbiology Laboratory Guidebook (MLG) and the U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) reference methods. E. coli O157:H7 ATCC 35150 and NM (non-motile) 700377 strains were used to inoculate the ground beef, beef trim, and spinach to obtain 20 low (0.23-2 CFU/test portion) and five high levels (3-5 CFU/test portion) of inoculations. Enriched samples were tested directly and subjected to anti-E. coli IMS prior to the SAS Molecular Tests. Results were determined via visual fluorescence and via turbidity using a turbidimeter. All the replicates, irrespective of the results, were confirmed using MLG 5.05 or BAM Chapter 4A methods. Results indicated that there were no significant differences in the detection of fractional positives (5-15 positives out of 20 replicates) with any of the methods tested above as compared to the reference methods. No false positives or negatives were detected except for two in the ground beef with IMS+Turbidity method. No false-negative samples were detected. Statistical analysis indicated that the modified methods were equivalent to the reference methods in detecting E. coli O157:H7 in the food matrixes tested. SAS Molecular Tests E. coli O157 Detection Kit can be used to detect E. coli O157:H7 in ground beef, beef trim, and spinach. The inclusion of IMS in the modified method improved the detection rate of E. coli O157:H7 in spinach and showed comparable detection rate in ground beef and beef trim. The optional use of visual fluorescent reagent and heat block either with IMS or without IMS produced results that were comparable to the results obtained from using a real-time lurbidimeter.

SAS molecular tests Escherichia coli O157 detection kit. Performance tested method 031203.

The SAS Molecular tests Escherichia coli O157 Detection method, a loop-mediated isothermal amplification method, performed as well as or better than the U.S. Department of Agriculture, Food Safety Inspection Service Microbiology Laboratory Guidebook and the U.S. Food and Drug Administration Bacteriological Analytical Manual reference methods for ground beef, beef trim, bagged mixed lettuce, and fresh spinach. Ground beef (30% fat, 25 g test portion) was validated for 7-8 h enrichment, leafy greens were validated in a 6-7 h enrichment, and ground beef (30% fat, 375 g composite test portion) and beef trim (375 g composite test portion) were validated in a 16-20 h enrichment. The method performance for meat and leafy green matrixes was also shown to be acceptable under conditions of co-enrichment with Salmonella. Thus, after a short co-enrichment step, ground beef, beef trim, lettuce, and spinach can be tested for both Salmonella and E. coli O157. The SAS Molecular tests Salmonella Detection Kit was validated using the same test portions as for the SAS Molecular tests E. coli O157 Detection Kit and those results are presented in a separate report. Inclusivity and exclusivity testing revealed no false negatives and no false positives among the 50 E. coli 0157 strains, including H7 and non-motile strains, and 30 non-E. coli O157 strains examined. Finally, the method was shown to be robust when variations to DNA extract hold time and DNA volume were varied. The method comparison and robustness data suggest a full 7 h enrichment time should be used for 25 g ground beef test portions.

SAS molecular tests Salmonella detection kit. Performance tested method 021202.

The SAS Molecular tests Salmonella Detection method, a Loop-mediated Isothermal Amplification method, performed as well as or better than the U.S. Department of Agriculture-Food Safety Inspection Service Microbiology Laboratory Guidebook and the U.S. Food and Drug Administration Bacteriological Analytical Manual reference methods for ground beef, beef trim, ground turkey, chicken carcass rinses, bagged mixed lettuce, and fresh spinach. The ground beef (30% fat, 25 g test portion), poultry matrixes and leafy greens were validated in a 6-7 h enrichment, and ground beef (30% fat, 375 g composite test portion) and beef trim (375 g composite test portion) were validated in a 16-20 h enrichment. The method performance for meat and leafy green matrixes was shown to be acceptable under conditions of co-enrichment with Escherichia coli 0157. Thus, after a short 6-7 h co-enrichment step, ground beef, beef trim, lettuce, and spinach can be tested for both Salmonella and E. coli O157. Inclusivity and exclusivity testing revealed no false negatives and no false positives among the 100 Salmonella serovars and 30 non-Salmonella species examined. The method was shown to be robust when enrichment time, DNA extract hold time, and DNA volume were varied.

Critical role of Bcl11b in suppressor function of T regulatory cells and prevention of inflammatory bowel disease.

Dysregulated CD4(+) T cell responses and alterations in T regulatory cells (T(reg) cells) play a critical role in autoimmune diseases, including inflammatory bowel disease (IBD). The current study demonstrates that removal of Bcl11b at the double-positive stage of T cell development or only in T(reg) cells causes IBD because of proinflammatory cytokine-producing CD4(+) T cells infiltrating the colon. Provision of WT T(reg) cells prevented IBD, demonstrating that alterations in T(reg) cells are responsible for the disease. Furthermore, Bcl11b-deficient T(reg) cells had reduced suppressor activity with altered gene expression profiles, including reduced expression of the genes encoding Foxp3 and IL-10, and up-regulation of genes encoding proinflammatory cytokines. Additionally, the absence of Bcl11b altered the induction of Foxp3 expression and reduced the generation of induced T(reg) cells (iT(reg) cells) after Tgf-ß treatment of conventional CD4(+) T cells. Bcl11b bound to Foxp3 and IL-10 promoters, as well as to critical conserved noncoding sequences within the Foxp3 and IL-10 loci, and mutating the Bcl11b binding site in the Foxp3 promoter reduced expression of a luciferase reporter gene. These experiments demonstrate that Bcl11b is indispensable for T(reg) suppressor function and for maintenance of optimal Foxp3 and IL-10 gene expression, as well as for the induction of Foxp3 expression in conventional CD4(+) T cells in response to Tgf-ß and generation of iT(reg) cells.