Development of a field-based assay for rapid detection of enterohemorrhagic Escherichia coli (EHEC).

摘要

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 (P0.005) 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.