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Operational Technologies Corporation (OpTech) proposes to further develop and optimize its already proven rapid, one-step fluorescence resonance energy transfer (FRET) DNA aptamer technology in a handheld sensor format to help detect and prevent the spread of agricultural bioterrorism. Advantages of the FRET-aptamer approach over conventional immunoassays include, possibly improved binding affinity and specificity over antibodies, one-step multiplexed assays, lowered cost versus antibody reagents, and freeze-drying of aptamer-FRET reagents for long-term storage with little or no loss of reactivity. OpTech has already demonstrated FRET-aptamer proof-of-concept for a bacterial (Shiga) toxin and Leishmania surface proteins under related projects. OpTech now proposes in Phase I to focus on a synthetic peptide representing a highly conserved region of an aphthovirus (foot-and-mouth disease; FMD) that has been targeted for virus neutralization. OpTech will clone and sequence the FMD FRET aptamers in Phase I and, if necessary, attempt to re-engineer physical placement of the fluorophore and quencher to optimize assay performance for maximal sensitivity and specificity based on the results of titration and cross-reactivity studies. In Phase II, the FRET-aptamer assay developed to detect the FMD peptide will be tested against whole, viable FMD virus particles in a commercially available handheld fluorometer.
NON-TECHNICAL SUMMARY: America is highly vulnerable to agricultural bioterrorism. A successful attack could be devastating to the U.S. and world economy. Rapid detection of agricultural bioterrorism agents is critical to limiting or halting the spread of such disease agents. This project concerns development of a rapid, facile, one-step fluorescence resonance energy transfer (FRET)-aptamer assay and handheld sensor for foot-and-mouth disease (FMD) viruses (possible agricultural bioterrorism agents). Re-introduction of FMD into the U.S. could be limited or stopped by appropriate quarantine measures if detected on farms early in the process of spreading. The handheld sensor and FRET assays are intended to fulfill this need. If successful, this platform technology could be developed to detect other bioterrorism agents as well.
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PROGRESS: 2006/05 TO 2007/12<BR>
During Phase I, OpTech developed and sequenced 10 candidate aptamers to a reportedly accessible (antibody neutralizable) and highly conserved 14-amino acid peptide from the surface of O-serotype FMD viruses. OpTech demonstrated promising FRET data using the PicofluorTM and polyclonal round 5 aptamers which was verifiable with a spectrofluorometer. However, the competitive FRET results were not nearly as robust when individual high-affinity and lower affinity aptamers were used for FRET. These results suggest that while the technology appears to work, further study and optimization are needed to enhance the sensitivity, specificity, and linear dynamic range of the competitive FRET-aptamer assay for FMD. One means by which to achieve these goals might be to make a cocktail of high and low affinity FMD aptamers that mimic the FRET results obtained with the polyclonal aptamers. If that was successful, the assays could be expanded to examine FRET from other FMD serotypes. Much was learned about the nature of OpTech's patent-pending competitive FRET-aptamer assay approach in this Phase I project. Difficulties with sequencing of convoluted GC-rich aptamer secondary structures were overcome by use optimization of the cloning and sequencing protocols. In addition, OpTech observed a dominant lights off FRET response from the polyclonal round 5 aptamer pool, but individual aptamers from that pool deomstrated either lights off or lights on FRET character and differences in sensitivity and specificity. The final assay may therefore consist of a cocktail of various known aptamer sequences selected to cover a specific dynamic range of analyte (FMD viral) concentrations and exclude certain known interfering species. The competitive FRET-aptamer assay approach does appear feasible for field-based detection of a variety of analytes in a handheld (Picofluor) format based on the polyclonal FRET-aptamer assay results. However, the latter work illustrates that, while OpTech is now proficient at obtaining DNA aptamer sequences, much more aptamer screening and optimization would be required to field a truly sensitive and specific FMD FRET-aptamer handheld assay system.
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IMPACT: 2006/05 TO 2007/12 <BR>
Although there has not been an outbreak of foot-and-mouth disease (FMD) in the U.S. since 1929 and all of North America is deemed FMD-free, the 2001 and 2007 outbreaks in the U.K. and other outbreaks in Asia remind us to be vigilant in monitoring for FMD. Unfortunately, no truly reliable field tests exist for all of the FMD serotypes. This fact necessitates sending of samples to the Plum Island Animal Disease Center which can isolate and identify an FMD serotype in as little as 4 days (USDA APHIS factsheet, Jan. 2002). Yet, even with quarantine procedures in place, the devastating aphthovirus can spread like wildfire over large distances and greatly damage the $100 billion U.S. livestock industry. To address the problem of a very rapid field test for FMD, the USDA awarded OpTech a Phase I SBIR contract to develop fluorescence resonance energy transfer (FRET)-DNA aptamer (surrogate antibody) homogeneous (one step, "lights on or off") assays that can be used to detect FMD in a plastic cuvette with a commercially available handheld, battery-operated fluorometer (the Picofluor by Turner Biosystems, Inc.).