Dr. Elizabeth A. Kearns Profile

Dr. Elizabeth A. Kearns

at Univ of South Florida

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Proceedings Article | 19 February 2009 Paper

Detection of E. coli O157:H7 in complex matrices under varying flow parameters with a robotic fluorometric assay system

Stephaney Leskinen, Sarah Schlemmer, Elizabeth Kearns, Daniel Lim

Proceedings Volume 7167, 71670J (2009) https://doi.org/10.1117/12.807830

KEYWORDS: Waveguides, Matrices, Biosensors, Robotics, Pathogens, Statistical analysis, Luminescence, Safety, Robotic systems, Radon

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The development of rapid assays for detection of microbial pathogens in complex matrices is needed to protect public health due to continued outbreaks of disease from contaminated foods and water. An Escherichia coli O157:H7 detection assay was designed using a robotic, fluorometric assay system. The system integrates optics, fluidics, robotics and software for the detection of foodborne pathogens or toxins in as many as four samples simultaneously. It utilizes disposable fiber optic waveguides coated with biotinylated antibodies for capture of target analytes from complex sample matrices. Computer-controlled rotation of sample cups allows complete contact between the sample and the waveguide. Detection occurs via binding of a fluorophore-labeled antibody to the captured target, which leads to an increase in the fluorescence signal. Assays are completed within twenty-five minutes. Sample matrices included buffer, retentate (material recovered from the filter of the Automated Concentration System (ACS) following hollow fiber ultrafiltration), spinach wash and ground beef. The matrices were spiked with E. coli O157:H7 (10³-10⁵ cells/ml) and the limits of detection were determined. The effect of sample rotation on assay sensitivity was also examined. Rotation parameters for each sample matrix included 10 ml with rotation, 5 ml with rotation and 0.1 ml without rotation. Detection occurred at 104 cells/ml in buffer and spinach wash and at 10⁵ cells/ml in retentate and ground beef. Detection was greater for rotated samples in each matrix except ground beef. Enhanced detection of E. coli from large, rotated volumes of complex matrices was confirmed.

Proceedings Article | 19 February 2009 Paper

Sample preparation and assay refinements for pathogen detection platforms

Daniel Lim, Elizabeth Kearns, Stephaney Leskinen, Sonia Magaña, Joyce Stroot, Dawn Hunter, Sarah Schlemmer

Proceedings Volume 7167, 71670O (2009) https://doi.org/10.1117/12.807763

KEYWORDS: Biosensors, Pathogens, Target detection, Fiber optics, Microorganisms, Biological research, Bacteria, Statistical analysis, Water, Luminescence

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Food-borne and waterborne microbial pathogens are a potential problem in biowarfare and public health. Such pathogens can affect the health, combat readiness, and effectiveness of the warfighter in a battlefield environment and present potential threats to the civilian population through intentional or natural contamination of food and water. Conventional procedures to detect and identify microbial pathogens in food, water, and other materials can take days to perform and may provide inconclusive information. Research at the University of South Florida's Advanced Biosensors Laboratory (ABL) focuses on development of sample processing procedures and biosensor-based assays for rapid detection of biothreat agents. Rapid processing methods, including use of an automated concentrator of microorganisms in water, have been developed for complex matrix samples including ground beef, apple juice, produce, potable water and recreational water, enabling such samples to be directly tested by biosensor assays for target analytes. Bacillus atrophaeus spores and other bacteria can be concentrated from potable and recreational water at low levels with a dead-end hollow-fiber ultrafiltration concentration system. Target bacteria recovered by these processing procedures can be identified by evanescent wave, fiber optic biosensors or other detection platforms. Fiber optic biosensor assays have been improved to include subsequent PCR analysis and viability determination of captured target bacteria using broth enrichment and/or ATP luminescence.

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