Detection of verotoxigenic Escherichia coli by magnetic capture- hybrization PCR

Abstract

Magnetic capture-hybridization PCR (MCH-PCR) was used for the detection of 36 verotoxigenic (verotoxin [VT]-producing) Escherichia coli (VTEC), 5 VTEC reference, and 13 non-VTEC control cultures. The detection system employs biotin-labeled probes to capture the DNA segments that contain specific regions of the genes for VT1 and VT2 by DNA-DNA hybridization. The hybirds formed were isolated by streptavidin-coated magnetic beads which were collected by a magnetic particle separator and, subsequently, amplified directly by conventional PCR. The detection system was found to be specific for VTEC: no amplification was obtained from non-VTEC controls, whereas VTEC isolates tested positive for one or two specific PCR products. With 5, 7, or 10 h of enrichment, the limits of detections were 103, 102, and 100 CFU/ml, respectively, by agarose gel electrophoresis. Southern hybridization did not seem to improve the limit of the detection. When applied to food, MCH-PCR was capable of detecting 100 CFU of VTEC per g of ground beef with 15 h of nonselective enrichment. The result of MCH-PCR for pure cultures of VT1-and/or VT2-producing E. coli cells were in total agreement with toxin production as measured by VT enzyme-linked immunosorbent assay.