| Rapid diagnostic assays for the detection of enterohemorrhagic Escherichia coli (EHEC) were developed and/or evaluated for use in field-based settings. These assays included: (1) three commercially available shiga toxin enzyme immunoassays, (2) a swab-based method (termed the Phast Swab) based on a T4 reporter bacteriophage (phage) that had been modified to carry a beta-galactosidase (lacZ) gene, and (3) another swab-based assay utilizing T-even phages that had been chemically labeled with enzymes (Phazymes). Each assay was evaluated for its ability to detect EHEC in pure culture and in food or food-related matrices. The specificity of the three enzyme immunoassays (Premier EHEC test [Meridian Bioscience Inc., Cincinnati, OH], the ProSpecT Shiga toxin E. coli STEC Microplate Assay [Remel Inc., Lenexa, KS], and the Ridascreen Verotoxin Enzyme Immunoassay [r-Biopharm AG, Darmstadt, Germany]) were not significantly different from one another (P>0.9) and showed high agreement with PCR for shiga toxin genes. The sensitivity of the Premier EHEC and ProSpecT STEC immunoassays were significantly better (P<0.005) than the Ridascreen Verotoxin immunoassay. The Premier EHEC immunoassay was able to detect 10 2 CFU/g EHEC in bovine feces following a 20-hour enrichment step.;The Phast Swab was evaluated for its ability to detect E. coli O157:H7 on meat samples. The assay was capable of detecting 10 2 CFU/100 cm2 of E. coli O157:H7 within 12 hours when a colorimetric substrate was used and 101 CFU/100 cm2 within 10 hours with the use of a luminescent substrate. However, the T4 lacZ reporter phage had a limited host range and was unhealthy due to the inactivation of its uvsY gene. This caused the reporter phage to have a slower infection time and lower burst sizes upon lysis, neither ideal for a rapid diagnostic. The Phazyme assay was evaluated for its ability to detect EHEC in broth trials. This assay was able to detect 100 CFU of EHEC within 9 hours when a luminescent substrate was used and 10 1 CFU within the same time frame with a colorimetric substrate for four of the five main EHEC serogroups. The Phazyme assay showed specificity greater than 93.1% for three of the five serogroups. When tested in food trials, the O157 Phazyme assay was able to detect E. coli O157:H7 in spinach consistently at levels of 1 CFU/g and occasionally at levels of 0.1 CFU/g, 102 CFU/100 ml in water samples, and 100 CFU/100 cm2 on swabbed meat samples. The Phazyme assay effectively detects EHEC in a simple and rapid manner, with minimal need for instrumentation to interpret the test result. |
