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United States Department of Agriculture

Agricultural Research Service

Title: Detection of Escherichia Coli 0157:h7 Through the Formation of Sandwiched Complexes with Immunomagnetic and Fluorescent Beads

Authors
item Tu, Shu-I
item Golden, Marsha
item Cooke, Peter
item Luce, Lauren - POLYSCIENCIES
item Paoli, George
item Gehring, Andrew

Submitted to: Journal of Rapid Methods and Automation in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: September 30, 2005
Publication Date: November 5, 2005
Citation: Tu, S., Golden, M., Cooke, P.H., Luce, L., Paoli, G., Gehring, A.G.2005 Detection of escherichia coli 0157:h7 through the formation of sandwiched complexes with immunomagnetic and fluorescent beads. Journal of Rapid Methods and Automation in Microbiology. 13:269-282

Interpretive Summary: Contamination of pathogenic bacteria, e.g., E coli O157:H7 in foods may lead to serious public health concerns. Thus, there is a need to rapidly, sensitively and specifically detect the pathogen in foods. For this purpose, immunomagnetic beads (IMB) have become a method of choice in many research laboratories. IMB can capture target pathogenic bacteria with specific antibodies on the beads and simple magnets can easily concentrate captured bacteria. The captured pathogen can then be detected using bio-recognition approaches (biosensors). However, the biosensor approach usually requires high-cost reagents. In a current study, we developed a new method that utilized a second immuno fluorescent bead (IFB) to reveal IMB-captured pathogen. We found that the new approach had similar detection sensitivity as other biosensor methods, e.g., time-resolved fluorescence. However, the cost of IFB approach was considerably lower since it is a common material routinely used for microsphere size calibration. The information is valuable for food safety laboratories to develop practical, automated and bead-based methodologies to detect pathogenic bacteria in foods.

Technical Abstract: A new fluorescent sandwich method for the detection of Escherichia coli O157:H7 in ground beef was developed. Immunomagnetic beads (IMB) pre-coated with anti-E. coli O157 antibody were used to capture and concentrate E.coli O157:H7 present in ground beef. Streptavidin-coated fluorescent beads (SFB) coated with anti-E. coli O157:H7 to form immunofluorescent beads (IFB) were used to detect the E.coli O157 captured by the IMB by forming IMBM-E. coli-O157:H7N-IFBO sandwich complexes where the subscripts M, N and O were variable but integral numbers. This sandwich technique was able to detect 500 CFU of the bacteria in a 0.5 mL sample taken from a quantified and diluted broth culture. The method was then applied to detect E. coli O157:H7 artificially contaminated in ground beef. Known quantities of freshly cultured E. coli O157:H7 cells were added to 25 g patties of ground beef held in sterile bags with filters. The inoculated patties were stored at 7 degrees C overnight; 75 mL of EC broth was added to each sample and the bags were incubated at 40 degrees C and 160 rpm for 4.5 hours. After the enrichment, E. coli O157:H7 in the filtrate were captured and concentrated with IMB and further conjugated with IFB for detection. The results demonstrated that the developed procedure can detect approximately 1.0 CFU of E. coli O157:H7 per gram of ground beef after a 4.5 hour enrichment period.

Last Modified: 7/25/2014
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