An official website of the United States government.
<OL> <LI> Develop and test a high-throughput strategy for the low-level detection of bacteria and other pathogens critical to food and water safety. <LI>Develop and refine a labeling scheme that provides redundancy in the verification of specific bacteria. <LI>Optimize magnetic separations to isolate target bacteria from complex samples. <LI>Adapt low-cost, state-of-the-art optical detection and electronic hardware for bacteria enumeration at the single microorganism level. <LI>Devise a two stage method for labeling bacteria by using fluorescent dyes that bind to the DNA of all cells in a sample and employing fluorescent compounds that bind specifically to the surface of target bacteria. <LI>Explore the use of aptamers as selective labels.
NON-TECHNICAL SUMMARY: A pathogen labeling strategy and multiplexed flow cytometry analysis system will be developed for high-throughput sampling and low-level detection of bacteria critical to food and water safety. The system will integrate advances in molecular recognition ligands, fluorescent labeling, magnetic separation, and high sensitivity optical array detection. Co-localization will be employed as a screening process that requires the presence of both dyes as confirmation for the positive presence of the target, eliminating problems associated with single-mechanism labeling techniques (false positives). Two labeling methods will be utilized; cell permeant dyes to label the DNA of all cells in the sample and fluorescently-labeled ligands that bind specifically to receptor sites on the outer surface of the target pathogen (antibodies and aptamers). This development will be combined with the optimization of magnetic separations for isolation of target pathogens from complex sample media and adaptation of low-cost, state-of-the-art optical, electronic. and detection hardware for pathogen enumeration at the single cell level without requiring enrichment.
<p>
APPROACH: This project focuses on the development and performance testing of a high-throughput strategy for the low-level detection of bacteria and other pathogens critical to food and water safety. The concept couples advances in selective labels, magnetic separations, and optical detection, The project has three parts: 1) development and refinement of a labeling scheme that provides redundancy in the verification of specific bacteria; 2) optimization of magnetic separations to isolate target bacteria from complex samples; and 3) adaptation of low-cost, state-of-the-art optical detection and electronic hardware for bacteria enumeration at the single microorganism level. The cornerstone of the concept is the simultaneous use of two distinct bacteria labeling mechanisms. One mechanism will use fluorescent dyes that bind to the DNA of all cells in the sample. The second mechanism will employ fluorescent compounds that bind specifically to the surface of target bacteria. The project also explores the use of a new class of selective labels known as aptamers, which have the potential to overcome the limitations of antibody-based labels. Overall, it is the effective integration of all these advances that will be critical to achieving the goals of the project.