The Applied Biosystems™ RapidFinder™ STEC Screening Assay is part of a complete, validated screening and confirmation workflow enabling TaqMan™ real-time PCR detection of the Shiga toxin-producing E. coli (STEC) O157:H7 and Big 6 non‐O157:H7 serogroup (O26, O45, O103, O111, O121 and O145) in a variety of beef products. The RapidFinder STEC Screening Assay was co-developed with the USDA-ARS and contains 96 complete qPCR assays targeted to screen for Shiga toxin 1 and 2 (stx1/stx2), eae, and potential O157:H7 presence. The assay is designed to be inclusive of all know allelic variants of stx and eae, including stx2g and stx2f. All reactions include an internal positive control (IPC) and ROX™ passive reference. Samples assessed as negative require no further testing. Samples assessed as positive must be further tested using the RapidFinder™ STEC Confirmation Assay.
The RapidFinder STEC Detection Workflow provides: • Greater confidence in results—part of a validated workflow for detection of as little as 1-5 CFU of pathogenic STEC in your sample • Simplified workflow—simultaneous detection of E. coli O157:H7 and the Big 6 non-O157:H7 strains in 375 g beef samples with a closed-tube, single-step lyophilized detection format that fits within your existing testing program • Reduced chance of false results—through use of high-quality DNA preparation, proprietary assay design, and internal positive control • Same day detection of pathogenic STECs and clearance of STEC-negative beef samples in as little as 12 hours, supporting early product release • Minimized costs and laboratory space with the use a lower volume of a nonproprietary single-step enrichment medium, eliminating the need to purchase proprietary media • Compatibility with RapidFinder™ Express software v.1.2 and later—enables easy, automated presence/absence detection and interpretation of results
Studies have shown that the RapidFinder STEC assays have a high level of sensitivity and specificity for E. coli O157:H7 and the Big 6 non-O157 STEC strains*. They detected all inclusion strains and showed no cross-reactivity to any of the exclusion strains tested. The stx assays detected all known variants of stx1 and stx2, including stx2f and stx2g. The optimized workflow co-developed with USDA-ARS showed equivalent detection to the MLG reference method, and the internal positive control in both assays significantly reduces the risk of false-negatives.
Applications The RapidFinder STEC Detection Workflow is intended for use by microbiological analysts who need to test for STEC in beef samples. Combined with the Applied Biosystems 7500 Fast RT-PCR System, RapidFinder STEC screening and confirmation assays provide a comprehensive solution for rapid detection, identification, and confirmation of Big 6 STECs and E. coli 0157:H7 to meet importation and exportation requirements for the beef industry. Specific workflows are detailed in the figures below and include both automated and spin column-based DNA isolation and real-time PCR detection of E. coli O157:H7 and Big 6 non-O157 strains in beef samples.
RapidFinder Express Software When integrated into the RapidFinder STEC Detection Workflow, RapidFinder Express Software automates data interpretation and generates user instructions on how to proceed with a sample. Data displays are available for novice and advanced users, showing simple presence/absence calls or raw data files using 7500 Fast System SDS Software.
RapidFinder STEC Screening and Confirmation Assays meet USDA FSIS guidance The RapidFinder STEC Detection Workflow, a two-stage real-time PCR method, is designed to accurately detect, identify, and confirm E. coli O157:H7 and the Big 6 serogroups of non-O157 Shiga toxin-producing E. coli defined by the USDA Food Safety and Inspection Service (FSIS) as adulterants in the American beef industry.