Author
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Brewster, Jeffrey |
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Submitted to: Journal of Microbiological Methods
Publication Type: Peer Reviewed Journal Publication Acceptance Date: 4/29/2003 Publication Date: 10/1/2003 Citation: Brewster, J.D. 2003. Isolation and concentration of salmonellae with an immunoaffinity column. Journal of Microbiological Methods. 55:28-293 Interpretive Summary: Rapid, sensitive assays capable of detecting small numbers of pathogenic bacteria in foods are needed by regulatory inspection personnel and others in agriculture and food industries working to reduce foodborne illnesses, such as those caused by Salmonellae. Current techniques are not able to detect very low levels of pathogens, so it is necessary to culture samples for periods of time to allow the pathogen to grow and increase in concentration before detection. This enrichment process adds hours to the assay, and may fail if the sample contains other benign microorganisms which grow more rapidly than the pathogen. To overcome this problem, a new method for concentrating pathogens in food samples has been developed. Small particles coated with antibodies against the pathogen (similar to those naturally produced in the body to attack bacteria) are packed into a tube or column, and the sample is allowed to flow over the particles. The pathogens stick to the antibodies, while the rest of the sample passes out of the column. By concentrating the pathogens into a small volume (less than a drop of water), this process serves the same purpose as enrichment. It is much faster than enrichment, and also separates the pathogens from any sample constituents that might interfere with detection. Future research will link this process to a number of existing detection methods to produce rapid, sensitive pathogen assays. Technical Abstract: A method for selectively isolating Salmonella from ground beef and concentrating the bacteria by a factor of approximately 500 was developed. Anti-Salmonella antibody was covalently linked to 40 micrometer polyacrylamide particles to prepare a solid phase with affinity for the bacteria. The particles were packed into 1 mm diameter glass tubes to form ma column 20 micro L in volume. Buffer and filtered ground beef extract containing Salmonella at concentrations ranging from 100 to 1 million /mL were pumped through the column to trap and concentrate the bacteria. At a flow rate of 200 micro L/min more than 95 percent of the bacteria introduced to the column were captured, while at 800 micro L/min capture dropped to 30 percent. Specificity was high, with no detectable capture of 100,000 /ml E. coli. By recirculating the sample through the column at a flow rate of 500 micro L/mL, over 90 percent of the Salmonella in a 5 mL sample were captured in 40 min. |
