title: Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay.
abstract: Conventional culture-based methods for detection of E. coli O157:H7 in foods and water sources are time-consuming, and results can be ambiguous, requiring further confirmation by biochemical testing and PCR. A rapid immunoassay prior to cultivation to identify presumptive positive sample would save considerable time and resources. Immunomagnetic separation (IMS) techniques are routinely used for isolation of E. coli O157:H7 from enriched food and water samples, typically in conjunction with cultural detection followed by biochemical and serological confirmation. In this study, we developed a new method that combines IMS with fluorescence immunoassay, termed immunomagnetic fluorescence assay (IMFA), for the detection of E. coli O157:H7. E. coli O157:H7 cells were first captured by anti-O157 antibody-coated magnetic beads and then recognized by a fluorescent detector antibody, forming an immunosandwich complex. This complex was subsequently dissociated for measurement of fluorescence intensity with Signalyte™-II spectrofluorometer. Experiments were conducted to evaluate both linearity and sensitivity of the assay. Capture efficiencies were greater than 98%, as determined by cultural plating and quantitative real-time PCR, when cell concentrations were <10(5) cells/mL. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (10-10(4) cells/mL), the fluorescence intensity of dissociated Cy5 solution was highly correlated with E. coli 157:H7 cell concentrations. The detection limit was 10 CFU per mL of water. The assay can be completed in less than 3 h since enrichment is not required, as compared to existing techniques that typically require a 24 h incubation for pre-enrichment, followed by confirmatory tests.
authors: Creatv MicroTech, Inc., Potomac, MD 20854, USA. pzhu@creatvmicrotech.com
authors: Peixuan
authors: P
authors: Zhu
authors: Daniel R
authors: DR
authors: Shelton
authors: Shuhong
authors: S
authors: Li
authors: Daniel L
authors: DL
authors: Adams
authors: Jeffrey S
authors: JS
authors: Karns
authors: Platte
authors: P
authors: Amstutz
authors: Cha-Mei
authors: CM
authors: Tang
mesh_terms: D058491:Bacterial Load / Q000295:instrumentation*; D015374:Biosensing Techniques / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D019453:Escherichia coli O157 / Q000302:isolation & purification*; D015200:Fluoroimmunoassay / Q000295:instrumentation*; D018189:Immunomagnetic Separation / Q000295:instrumentation*