Grant: $2,727,684 - National Institutes of Health - Aug. 14, 2009
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Award Description: In order to protect civilian and military populations, it is critical to improve the diagnosis of NIAID Category A-C priority pathogens. More rapid, more sensitive, more specific, and more informative diagnosis than currently possible will allow earlier and targeted treatment of the individual patient, improved strategic and tactical responses by civilian and military authorities, and more effective remediation activities. The goal of this research program is to develop a prototype instrument that will allow the rapid, sensitive, and specific identification NIAID Priority Pathogens in clinical samples by nucleic acid sequencing. The prototype system will identify three pathogens, B. anthracis (vegetative cells and spores), Y. pestis, and B. pseudomallei; additional Category A-C Priority Pathogens will be added following successful completion of the two-year program. In addition, the technology developed over the course of this research will be directly applicable to the development of clinical diagnostics for common infectious diseases. The prototype system will generate approximately 500 bp of QV20 sequence from each of eight loci per biothreat—a number of loci large enough to provide strain-specific biothreat identification but small enough to allow generation of results within one hour of clinical sample collection. In contrast to other detection technologies, the proposed sequencing system will allow identification of closely related species and strains; sequencing will allow biothreats to be distinguished from non-pathogenic near neighbors. The system in development consist of three components: (1) A Smart Cartridge (SC) will accept a clinical sample (e.g. blood), and extract and purify DNA; (2) An Integrated Biochip (IB) will perform highly multiplexed amplification, Sanger sequencing, and electrophoretic separation and laser-induced detection; (3) A ruggedized fully integrated instrument will accept the SC and IB and performs all required process steps. The proposed program is focused on the following activities: (1) Design and testing of biothreat primers and development of analytic software (2) Design, fabrication, and testing of a SC that incorporates Network Biosystem’s existing cell separation and microfluidic DNA purification technologies with a spore lysis system to be developed at NetBio; (3) Design, fabrication, and testing of an IB that integrates microfluidic amplification, sequencing, and electrophoretic separation and detection; and (4) Testing of the SC, IB, and instrument using model systems. The prototype will allow demonstrations of the system to validate the rapid biothreat sequencing approach. The proposed research is intended to challenge the current clinical paradigm by providing a rapid biothreat sequencing system that will meet the following objectives: • Sensitivity and specificity. The proposed system will allow definitive diagnosis soon after initial symptoms become apparent. Generation of sequence data from several loci per biothreat will minimize false positives. • Time to answer. DNA sequence identification will be provided one hour following sample introduction, allowing prompt initiation of specific treatment to improve patient outcomes and appropriate defensive and remediation measures to improve societal outcomes. • Ease-of-use. Operator functions will be limited to inserting sample into a tube and pressing a start button. • Closed system. The system will be self-contained to prevent contamination and errors in sample handling and to protect the operator. • Site of Operation. The instrument will be ruggedized for use in a central testing facility, local hospital, or in the field. Conducting the diagnostic test at the point of care will further reduce time to diagnosis. In summary, the proposed protoype system will allow much more detailed information about biothreats to be obtained much more quickly than is currently possible.
Project Description: Spores and Ultrasonication: A major goal of the program is to sequence DNA from biothreats that can speculate. B. anthracis produce spores that are extremely resistant to conventional lysis techniques. Accordingly, we have initiated work on an ultrasonic module for spore lysis. An excellent non-pathogenic model for testing B. anthracis lysis is B. licheniformis, which produces an exosporium that is essentially identical to that of anthrax itself. Experiments to cultivate and induce sporulation in B. licheniformis have been completed, and a series of assays to quantify spore number and ensure that all vegetative forms and free DNA have been removed is in progress. These characterization experiments are critical to enable an accurate assessment of the quantitative effect of sonication. In parallel, several basis approaches to ultrasonication are being evaluated. At present, the initial studies are testing the ability of commercial off-the-shelf systems to generate the power estimated to be required for lysis. A series of tools are being developed to assess the coupling between a given ultrasonic horn and the sample-containing biochip as measured by the heat generated and the amplitude of the ultrasonic pressure waves in the liquid. Microfluidics: Two major components are required for the prototype system, a Smart Cartridge and an Integrated Biochip. The Smart Cartridge was designed and fabricated, and a quality control system to allow flow in microfluidic channels to be quantified was completed. This is particularly important, as CNC-machining and bonding methods used in development are not as reproducible as large scale manufacturing methods such as injection molding. In addition, several methods for membrane placement in the Smart Cartridge were designed and tested, and an optimal design was selected. Based on these advances, design of the first iteration biochip has been initiated.
Jobs Summary: The 6 FTEs functions that were retained as a result of this award ARRA program are 2 research associates, 2.5 scientists, .5 engineer, and 1 program management. (Total jobs reported: 6)
Project Status: Less Than 50% Completed
This award's data was last updated on Aug. 14, 2009. Help expand these official descriptions using the wiki below.
Funds from this award have been disbursed to sub-grantees. Click here to see a list of sub-grantees.
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