Bacterial Sensors for Food Safety

NON-TECHNICAL SUMMARY: Ensuring the safety of our food supply from natural or deliberate acts of contamination is a national priority that affects all citizens of these United States. The development of new methodologies that can rapidly detect the presence of toxins and pathogenic bacteria in food products and trace the tainted food back to its origin must be a part of any comprehensive prevention strategy to lower the incidence of food-borne illness. This research project is part of a ten year strategic plan to develop Radio-Frequency Identification (RFID) sensor tags for the rapid detection of food-borne bacteria such as Salmonella typhimurium. In response to recent concerns of agro-terrorism, this research is pursuing the development of detection technologies that can be used to rapidly identify deliberate contamination of foods with bio-threat agents such as anthrax, Salmonellae and ricin. New phage-based technologies with improved detection limits and shelf-lives are being investigated. The long range goal is to incorporate these sensors as part of the RFID sensor tags to enable rapid detection of an agro-terrorism attack.

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APPROACH: 3.0 PROCEDURES 3.1 Experiments The work to be conducted during this year of the project will be divided into two Tasks, Task A: Commercialization of Phage Based Test Kits and Task B: Miniaturized Sensor Platforms. Research under Task A: Commercialization of Phage Based Test Kits will focus on the development of phage based ELISA test strips for the detection of Bacillus anthracis spores and Salmonella typhimurium cells. Commercialization is anticipated to require 24 months of research. The development of these test strips was made possible by the highly successful research conducted last year under this USDA grant that demonstrated the superior properties of phage based bio-molecular recognition versus antibody based bio-molecular recognition. Research under Task B: Miniaturized Sensor Platforms will continue investigations of the highly successful magnetostrictive particle sensors and other sensor platforms that will serve as transduction devices in the detection of small amounts of bacteria, spores and toxins. Task A research will be subdivided into two subtasks. Subtask A.1: Phage based test kits for the detection of Bacillus anthracis and Subtask A.2: Phage based test kits for the detection of Salmonella

<P>PROGRESS: 2006/06 TO 2007/06<br>
Auburn University through this multi-year research project is developing new methods of rapid pathogen detection. The research that was conducted this year was divided into two Tasks (Task A: Phage Based Test Kits and Task B: Miniaturized Sensor Platforms). Research under Task A focused on the development of phage based ELISA test strips for the detection of Bacillus anthracis. Task A research discovered that salt concentration in the solutions used to introduce the phage to the sensor test strip or sensor platform controls the phage distribution and hence binding affinity of phage. Transmission electron microscopy found that for low salt concentration solutions the phages aligned and formed bundles. As the salt concentration was increased there was an optimal salt concentration where phage bundles disassociated into individual phage elements. At very high salt concentrations, the phages again formed bundles. Affinity binding tests were conducted in the different salt concentration solutions and binding was measured to be the greatest for conditions where the phage disassociated into individual elements. To investigate the effect of phage bundle formation on platform biosensors, different salt concentration solutions were used to introduce the phage to the biosensor platform surface. Scanning electron microscopy of the biosensor platform surface showed greatly improved binding of the target organism occurred for conditions of individual immobilized phages. A second research effort being conducted under Task A is the improvement of the specificity of the phage used as the biomolecular recognition element in both ELISA based test strips and platform biosensors. As an initial step, we constructed a landscape phage library via a method that minimized Escherichia coli codon bias to enhance translation of rare codons. In addition, we improved biopanning procedures and isolated oligopeptides from two different phage display libraries (pIII-displayed 12-mer oligopeptides on M13 phage and pVIII-displayed 8-mer oligopeptides in landscape conformation on fd-1 phage) that demonstrated high specificity to B. anthracis spores. Initial data indicate that the phage clone RA13, isolated from the pIII library, demonstrates higher specificity to B. anthracis spores than our previously developed JRB7, a landscape phage with high affinity for B. anthracis spores. From the pVIII landscape library, we isolated several classes of phage clones whose sequences were different from that of JRB7. We are currently testing the specificity of these phage clones to determine their specificity and affinity for B. anthracis spores compared to JRB7. Research under Task B (Miniaturized Sensor Platforms) demonstrated that magnetoelastic particle sensors can be fabricated with dimensions of 50 nanometers in diameter by 1000 nanometers long. This size of sensor is capable of detecting the attachment of a single spore or bacteria. Difficulties were experienced in measuring the characteristic resonance frequency of these nanosize biosensors. Research is continuing to improve resonance frequency measurement capabilities.
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IMPACT: 2006/06 TO 2007/06<BR>
Over 76 million Americans become ill each year due to foodborne illness. These foodborne illnesses will leave an estimated 9,000 Americans suffering from chronic complications and result in more than 5,000 deaths. Foodborne illness costs more than $30 billion annually in lost productivity. Ensuring the safety of our food supply from natural or deliberate acts of terrorism is therefore a top priority. Under this research project, sensors that will allow the rapid detection of pathogens (bacteria, spores and toxins) will be investigated. The sensors will be designed, fabricated and tested to determine their sensitivity, specificity and longevity both in control liquids and in food products. This project is part of a ten year plan to reduce foodborne illness and detect agroterrorism attacks by improving our ability to rapidly identify pathogens in real time under field conditions. With real-time sensors that are able to detect levels of pathogens that make a person sick, food that is contaminated can be removed from the food chain during harvesting or during initial stages of processing. This early identification of contaminated food will thereby eliminate cross-contamination and reduce the incidence of foodborne illness.