Developing Bead-Based Microarray for Detection of Shiga Toxin-Producing E. Coli (STEC)

Non-Technical Summary:<br/>
The CDC estimates STEC causes approximately 175,000 illnesses, 2,400 hospitalizations and 20 deaths annually in US alone. While O157 serogroup is most well-known, non-O157 serogroups, particularly those belonging to six O groups, O26, O111, O103, O121, O45, and O145, have been recognized as a growing public health concern. Since STEC infections can lead to serious complications such as hemorrhagic colitis, hemolytic uremic syndrome and even death, it is highly crucial to detect STEC infection to properly treat the patients and control the outbreaks in timely manner. In this study, we propose to develop a microbead-based suspension array for STEC detection and identification of its serogroup, which can be applied to various food and environmental samples. Currently there are no selective media that are able to identify non-O157 STEC. A sensitive non-culture method to detect all STEC strains with minimal cross-reactivity would be valuable to control STEC contamination in foods and STEC-associated infections in public. Moreover, when detection techniques are applied for pre-harvest food safety to control STEC contamination in cattle or fresh produce, a detection assay that could provide results within 24 hrs (compared to 3-4 days for culture methods) would be highly desirable in that it would allow producers time to implement corrective actions and control further transmission. This study proposes a unique approach in detecting multiple foodborne pathogens or multiple serotypes by employing microbead-based array. Microbead-based arrays can provide sensitivity specificity along with high degree of multiplexing ability. Compared to standard culture methods, bead-based array using pre-encoded microspheres can provide much improved rapidity and simplicity. Additionally, this study offers a unique approach by employing two different molecular diagnostics: immunosensor arrays and DNA-sensor arrays. Redundancy of bead sensors and their ability to simultaneously detect and characterize multiple pathogens will make them cost-effective and sensitive assay system for pathogen detection.
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Approach:<br/>
We will select genes for detection and identification of STEC, focusing on virulence genes, and design probes and primers using a probe/primer design software for array application. Whenever possible, multiple probes will be designed for each target gene to increase the specificity of the assay. All primers and probes will be synthesized with appropriate chemical modification for bead conjugation and covalently coupled to optically encoded, carboxylated microspheres. Each probe-coupled bead type will then be tested its performance using fluorescently labeled synthetic targets. Once each bead type is tested for its sensitivity and specificity, all probe-coupled beads will be combined together to create bead library and added to each well in microtiter plate to create a DNA sensor array. The developed multiplexed array will then be tested with synthetic targets to evaluate sensitivity and specificity. For this test, flow cytometry-based multiplexing array system will be used as a main platform. These systems can accommodate standard 96-well microplate, which is the format used for the proposed bead-based array. Each bead-probe set will be registered using two-laser detecting device (red and green). This registration method will eliminate the need to identify and register each bead, which is the common drawback of other bead-based arrays. Various concentrations of synthetic targets will be tested on the array either as a single sample or as a combination of multiple samples. To develop immunosensor array, the microbeads coated with antibodies against different serotypes of STEC will be utilized. Antibodies will be covalently coupled to carboxylated microspheres, and each bead type functionalized with antibody to STEC will be tested individually for its performance as described above. Heat-killed STEC cells that are commercially available will be used as test samples for this objective. Once the feasibility of the developed DNA-sensor microarray is confirmed with synthetic targets, it will be tested with DNA isolated from STEC culture. PCR amplification will be used for target labeling as well as amplification. For immunosensor microarray, culture of STEC will be used. Both microarrays (DNA sensor and immunosensor arrays) will be tested for their sensitivity with various concentrations of target cells. The developed arrays will also be tested for their specificity with other non-STEC foodborne pathogens to confirm if the arrays can selectively detect STECs without any cross reactivity. Once the sensitivity and specificity are confirmed for the developed arrays, the arrays will be applied to detect STEC in foods of complex matrices such as ground beef. Once the developed arrays can successfully detect STEC, the arrays will be modified by adding more beads to simultaneously detect other foodborne pathogens such as Salmonella, Listeria, Staphylococcus aureus, Campylobacter, and Shigella. We will also compare the sensitivity and specificity of the developed arrays for STEC detection with those of other conventional methods including culture-based method, PCR-based method and ELISA.
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Progress:<br/>
2012/07 TO 2012/09<br/>
OUTPUTS: Multiple target genes for detection of initial target organisms including Shiga toxin-producing E. coli (STEC) O157, O26, O45, O103, O111, O121, and O145 have been chosen: wzx gene for O26, O45, O103, O111, and O145; wbqE and wbqF genes for O121; rfb gene for O157 as well as four major STEC virulence genes (stx1, stx2, ehxA, and eae). Along with identified target genes, the clustered, regularly interspaced, short palindromic repeats (CRISPR) regions of STECs which show genetic diversity were also selected for further identification of STECs (Genbank accession number JX219753 to JX219760). Bioinformatics software for microarray application, AlleleID (Premier Biosoft, Palo Alto, CA) was employed to design probes and primers for all target genes. Using AlleleID, at least one probe for detection and a pair of primers for PCR amplification, and two probes and two sets of primers if possible, were designed for stx1, stx2, eae, ehxA and rfb target genes and probes and primers are currently being designed for other genes. Designed probe and primer sequences were checked against GenBank Database to ensure their specificity. It was noted that some of the selected target genes showed high sequence homology with Shigella spp or non-Shiga toxin-producing E. coli. To solve the problem, new probes and primers are currently being designed for these genes which can avoid or minimize cross-reactivity with non-target organisms or with other serotypes, and more genes are being explored to replace the genes with high sequence homology.
<br/>PARTICIPANTS: Soohyoun Ahn (PI), Food Science and Human Nutrition Dept., University of Florida Tyler Austin (student), Food Science and Human Nutrition Dept., University of Florida Shuang Wu (student), Food Science and Human Nutrition Dept., University of Florida David Gilmore (collaborator), Biological Sciences, Arkansas State University Donald Kennedy (collaborators), College of Agriculture and Technology, Arkansas State University.
<br/>TARGET AUDIENCES: This project will be benefit government agency and food producers and processing industry by providing means to test foodborne pathogens in food products to assure food safety. Additionally, this project will ultimately benefit general public by protecting public health.
<br/>PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
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IMPACT: Studies described above indicated that depending on virulence-related genes alone for simultaneous detection and serotype identification of multiple STECs might cause cross-reactivity problems. Since the target STECs share high similarity in their pathogenicity, it would be hard to identify each these pathogens at the serootype-specific level. Alternative approach to this problem will be identification of target using pattern-recognition rather than simple yes/no response. When detecting multiple pathogens which are close to each other phylogenetically, pattern-recognition approach may prove more useful for correct identification. Currently, the designed primers are being tested by multiplex PCR for detection and identification of STECs in environmental samples collected from cattle farms. When designed primers and probes are incorporated into multiplexing detection platform, which is the ultimate goal of this project, it will improve food safety and public health by identifying STECs in foods with rapidity and sensitivity.