Progress 10/01/11 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): The objective of the proposed collaboration is for USDA ARS and DuPont Qualicon to successfully develop real-time PCR assays for the detection of important non-O157 Shiga toxin producing E. coli (STEC), including serogroups O26, O45, O103, O111, O121, and O145. targeting virulence genes and also serogroup specific genes. Approach (from AD-416): This collaboration involves the inclusion of multiple aspects for the design and optimization of methods yielding successful detection of six serogroups of non-O157:H7 STECs, including O26, O45, O103, O111, O121, and O145. The first aspect provides focus on the effort to optimize enrichment methods used for successful propagation of all serogroups of interest. In regard to real-time PCR assay development, there will be two areas of focus. The first includes developmental efforts for the determination of the presence of Shiga toxin genes and the intimin gene (eae) as a screening method. The second area focuses on methods for the individual identification of each of the six non-O157:H7 serogroups listed above based on amplification of serogroup-specific genes. Upon optimization of the chemistry, inclusion of an internal positive control, and determination of grouping of serogroup specific primers and probes, the PCR reagents will be evaluated using reagents in the form of a tablet. Evaluation of the entire process of enrichment, detection of toxin and intimin genes, and identification of each STEC serogroup using the tableted PCR reagents, is the goal of this collaboration. A test kit consisting of rapid polymerase chain reaction-based screening assays for detection of the top six non-O157 STEC serogroups was developed and is currently being commercialized by the CRADA partner. Rapid screening methods that can be used by the food industry for detection of six non-O157 Shiga toxin-producing E. coli (STEC) serogroups that were declared as adulterants in beef by the FSIS are needed. The food industry cannot hold perishable food for long periods; therefore, rapid methods for detection of contamination with pathogens must be used. The assays developed by ARS researchers in Wyndmoor, PA and the CRADA partner can be used by regulatory agencies and the food industry to detect the presence of the six STEC serogroups in food. These assays allow screening of beef products for the six serogroups targeting important STEC genes that are carried by these pathogens. They accurately detected and identified the six non-O157 STEC strains, and they simplify testing for non-O157 STEC since they require fewer hands-on steps. The assay kit will be useful for testing for non-O157 STEC, particularly for the food industry in the U.S. and throughout the world, since the FSIS will require that beef that is imported into the U.S. be tested for the top six non-O157 STEC, and use of this detection kit will prevent expensive beef recalls by the industry.
Impacts
(N/A)
Publications
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Progress 03/01/10 to 09/30/12
Outputs
Progress Report Objectives (from AD-416): The objective of the proposed collaboration is for USDA ARS and DuPont Qualicon to successfully develop real-time PCR assays for the detection of important non-O157 Shiga toxin producing E. coli (STEC), including serogroups O26, O45, O103, O111, O121, and O145. targeting virulence genes and also serogroup specific genes. Approach (from AD-416): This collaboration involves the inclusion of multiple aspects for the design and optimization of methods yielding successful detection of six serogroups of non-O157:H7 STECs, including O26, O45, O103, O111, O121, and O145. The first aspect provides focus on the effort to optimize enrichment methods used for successful propagation of all serogroups of interest. In regard to real-time PCR assay development, there will be two areas of focus. The first includes developmental efforts for the determination of the presence of Shiga toxin genes and the intimin gene (eae) as a screening method. The second area focuses on methods for the individual identification of each of the six non-O157:H7 serogroups listed above based on amplification of serogroup-specific genes. Upon optimization of the chemistry, inclusion of an internal positive control, and determination of grouping of serogroup specific primers and probes, the PCR reagents will be evaluated using reagents in the form of a tablet. Evaluation of the entire process of enrichment, detection of toxin and intimin genes, and identification of each STEC serogroup using the tableted PCR reagents, is the goal of this collaboration. A test kit consisting of rapid polymerase chain reaction-based screening assays for detection of the top six non-O157 STEC serogroups was developed and is currently being commercialized by the CRADA partner. Rapid screening methods that can be used by the food industry for detection of six non-O157 Shiga toxin-producing E. coli (STEC) serogroups that were declared as adulterants in beef by the FSIS are needed. The food industry cannot hold perishable food for long periods; therefore, rapid methods for detection of contamination with pathogens must be used. The assays developed by ARS researchers in Wyndmoor, PA and the CRADA partner can be used by regulatory agencies and the food industry to detect the presence of the six STEC serogroups in food. These assays allow screening of beef products for the six serogroups targeting important STEC genes that are carried by these pathogens. They accurately detected and identified the six non-O157 STEC strains, and they simplify testing for non-O157 STEC since they require fewer hands-on steps. The assay kit will be useful for testing for non-O157 STEC, particularly for the food industry in the U.S. and throughout the world, since the FSIS will require that beef that is imported into the U.S. be tested for the top six non-O157 STEC, and use of this detection kit will prevent expensive beef recalls by the industry.
Impacts
(N/A)
Publications
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