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Title: Sensitive detection of E. coli O157:H7

Author
item ZHU, PEIXUAN - Creatv Micro Tech, Inc
item Shelton, Daniel
item ADAMS, DANIEL - Creatv Micro Tech, Inc
item LI, SHUHONG - Creatv Micro Tech, Inc
item TANG, CHA-MEI - Creatv Micro Tech, Inc
item AMSTUTZ, PLATTE - Creatv Micro Tech, Inc

Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 4/27/2009
Publication Date: 4/27/2009
Citation: Zhu, P., Shelton, D.R., Adams, D., Li, S., Tang, C., Amstutz, P.T. Sensitive detection of E. coli O157:H7. Meeting Abstract.

Interpretive Summary:

Technical Abstract: Introduction: Escherichia coli O157:H7, the most common serotype of enterohemorrhagic E. coli, is responsible for numerous food- and water-borne infections worldwide. 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 confirmation by biochemical testing or PCR. There is a need for rapid screening method that can identify presumptive positive sample prior to cultural plating to save time and resources. In this study, we have developed a new method by combining immunomagnetic separation (IMS) with fluorescence immunoassay (FIA) for the detection of water-borne E. coli O157:H7. Materials and methods: E. coli O157:H7 bacterial cells in spiked water samples were first captured on the surface of the magnetic beads coated with specific monoclonal anti-O157 antibodies. Other bacteria and contaminants were removed by subsequent washes. The captured E. coli O157:H7 bacteria were recognized by a polyclonal anti-O157 detector antibody labeled with Cy5 fluorescent dye. The immunosandwich complex on the magnetic beads was dissociated and detected in aqueous phase using an ultra sensitive Signalyte-II spectrofluorometer. Results and discussions: The capture efficiency of IMS was 100 percent for input E. coli O157:H7 concentrations of 10x2 cells, and 98.8 percent for 10x3 to 10x5 cells. Fluorescence immunoassay conditions have been optimized to reduce background noise and increase dissociation efficiency. A 10-fold serial dilution of E. coli O157:H7 ranging from 10 to 10x6 CFU/ml was analyzed by IMS in conjunction with fluorescence immunoassay (IMS-FIA). Based on fluorescence intensity from the negative controls, a threshold detection value was set at (average+3S.D). Samples containing E. coli O157:H7 concentration as low as 10 CFU/ml were able to generate Cy5 fluorescence signal higher than the threshold. Conclusions: Thus, the detection limit using IMS-FIA for E. coli O157:H7 was less than 10 CFU/ml with assay time less than 3h using Signalyte-II. This assay protocol can be adapted for the detection of other water- and food-borne pathogens.