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United States Department of Agriculture

Agricultural Research Service

Title: Detection of Immunomagnetically Captured Escherichia Coli O157:h7 by Antibody-Conjugated Alkaline Phosphatase

Authors
item TU, SHU-I
item Patterson, Deirdre
item Briggs, Constance
item IRWIN, PETER
item Yu, Linda

Submitted to: Journal of Industrial Microbiology and Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 1, 2001
Publication Date: N/A

Interpretive Summary: Contamination of pathogenic bacteria, e.g., E coli O157:H7 in foods may lead to serious public health concerns. To minimize possible outbreaks of food poisoning by this bacteria, sensitive and rapid detection techniques are needed to call for proper treatments to intervene further distribution of contaminated foods. Current available methods for detecting pathogenic bacteria are time consuming or of low specificity. In an attempt to develop specific and sensitive methods that utilize the 96-well micro-plate reader, a common instrument found in most microbiological laboratories, we designed a new process in which cells of E. coli O157:H7 were captured by forming sandwiched complexes with two specific antibodies coated on magnetic beads and linked to an enzyme (alkaline phosphatase), respectively. The sandwiched complexes were concentrated and separated from other components with a magnet. The enzyme in the complexes then generated intensely-colored compounds that could be quantified by the plate-reader. In application, hamburger spiked with about 1 cell of E. coli O157:H7 per gram of meat, showed positive response after 4 to 6 hours of enrichment. With this method, the presence of low levels of E. coli O157:H7 may be determined within 8 hours. Furthermore, the approach can meet the high throughput requirement by the use of existing 96-well micro- plate reader. The information is useful for researcher and/or engineers to design an automated process for detecting specific pathogens in foods.

Technical Abstract: A rapid and sensitive detection process for Escherichia coli O157:H7 was developed. Alkaline phosphatase-labeled anti-E. coli O157 antibodies were used to first tag the targeted bacteria. Immunomagnetic beads or antibody- labeled streptavidin-coated magnetic beads were then used to capture the APase tagged E. coli. A magnet was then used to concentrate and separate the bacteria away from other components. Immunomagnetically-captured bacteria were thereupon washed and distributed into micro-plates or optical cuvettes. The enzyme-catalyzed hydrolysis of p-nitro-phenol phosphate in alkaline solutions was then followed from the absorbance increase at approximately 400 nm. With pure cultures of E. coli O157:H7, less than 1000 CFU/mL could be detected. This approach was applied to detect the bacteria artificially spiked in beef hamburgers. Less than 1 CFU/g of E. coli O157:H7 produced a significant response after cultural enrichment for four to six hours at 37 oC. The presence of E. coli K-12 showed no effect on the detection of the target bacteria. Thus, this method may be used as a convenient screening process for E. coli O157:H7 in foods.

Last Modified: 7/28/2014
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