Comparative Performance Evaluation of Real-Time PCR and Dual-Labeled Fluorescence Resonance Energy Transfer Probe-Based Melt Peak Analysis for the Detection of Escherichia coli O157:H7 in Beef Products

Contaminated beef and beef products remain a frequent vehicle for the transmission of Escherichia coli O157:H7. The current U.S. Department of Agriculture (USDA) Food Safety and Inspection Service (FSIS) regulatory testing for E. coli O157: H7 uses the method described in the USDA-FSIS Microbiology Laboratory Guidebook (MLG), chapter 5. At times, described presumptive test results are nonconfirmable, suggesting that recent PCR technological advancements and presumed enhanced sensitivity and specificity may offer beneficial changes. Here, we have evaluated the precision and sensitivity of a fluorescence resonance energy transfer-based real-time PCR assay called ECO for the detection of E. coli O157:H7. ECO detects the gene target specific to both E. coli O157:H7 and E. coli O157:non-H7 but distinguishes the two by using a melt curve analysis. A total of 3,113 O157:H7 and O157:non-H7 isolates were used to define this melting temperature-based criteria. The simulated comparative performance evaluation in the spiked beef samples indicated detection of 3 of 3 samples by ECO at