PATHOGEN FATE AND TRANSPORT IN IRRIGATION WATERS
Location: Environmental Microbial and Food Safety Laboratory
Title: Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay
Submitted to: Biosensors and Bioelectronics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 22, 2011
Publication Date: December 15, 2011
Citation: Zhu, P., Shelton, D.R., Li, S., Adams, D., Karns, J.S., Amstutz, P., Tang, C. 2011. Detection of E. coli O157:H7 by immunomagnetic separation coupled with fluorescence immunoassay. Biosensors and Bioelectronics. 30:337-341.
Interpretive Summary: There is a pressing need for rapid, sensitive methods for detection of pathogens in food. One such bacterial pathogen is E. coli O157:H7, which can occur as a contaminant of beef or produce and is responsible for potentially life-threatening gastrointestinal illness. A variety of methods have been developed to detect E. coli O157:H7, including fluorescence assays. Fluorescence assays consist of combining bacterial cells with O157-specific antibodies labeled with fluorescent dyes (the fluorescent dyes “glow” when illuminated with UV light). Since the O157-specific antibodies recognize and attach only to O157 cells, the cells can be detected even when present at low concentrations in a mixture of other bacteria. Most fluorescence assays require several thousand cells for detection, so cells must grow (be enriched) prior to analysis. The current study describes a fluorescence assay that can detect as few as 10 cells due to the use of a very sensitive detector. Consequently, pre-enrichment is not required for detection which shortens the analysis time by several hours. This research will be of interest to other scientists, the food industry and public health laboratories.
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 SignalyteTM-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 <105 cells/ml. Capture efficiency decreased at higher cell concentrations, due to the limitation of bead binding capacity. At lower cell concentrations (104 to 101 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.