Detection of verotoxigenic Escherichia coli by magnetic capture-hybridization PCR.
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 hybrids 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 detection were 10(3), 10(2), and 10(6) 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 10(0) CFU of VTEC per g of ground beef with 15 h of nonselective enrichment. The results 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 a VT enzyme-linked immunosorbent assay.
Chen
J
J
Department of Food Science, University of Guelph, Ontario, Canada.
Johnson
R
R
Griffiths
M
M
D001427:Bacterial Toxins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D001710:Biotin / Q000378:metabolism; D015169:Colony Count, Microbial; D015342:DNA Probes / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005516:Food Microbiology; D005798:Genes, Bacterial; D008280:Magnetics; D009693:Nucleic Acid Hybridization / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D022622:Shiga Toxin 1; D019809:Streptavidin
D001427:Bacterial Toxins; D015342:DNA Probes; D004269:DNA, Bacterial; D022622:Shiga Toxin 1; D001710:Biotin; D019809:Streptavidin