1. Improving the Sensitivity and Reliability of Fluorescence Methods Used to Detect Biological Threats
The assays used to detect biological threats must be very sensitive and highly specific. Environmental samples are highly complex, heterogeneous, and contain a background of endogenous organisms. Detection methods must reliably detect threats from such samples. Fluorescence reporters have the necessary sensitivity and are coupled to molecular recognition compounds to provide necessary specificity. The molecular recognition compounds used include antibodies and nucleic acids. Antibody and nucleic acid–based assays offer flexibility and the specificity and speed for the detection of dangerous bacteria, viruses, and toxins. We are working to improve the sensitivity and reliability of these fluorescent-based assays for the detection of biological threats.
The immobilization of nucleic acids on surfaces in large numbers (microarrays) have produced a revolution in the speed and scale at which gene expression experiments are done in science. The nucleic acids from biological threat organisms are detected by measurement of the fluorescence intensity following hybridization of the nucleic acids to the microarrays. Thus, microarrays offer the potential for the rapid detection of a large number of biological threats in the environment. However, a number of significant technical barriers must be overcome before the use of microarrays for detection of biological threats becomes fully operational. Problems include the presence of materials in the samples that can cause false positives and false negatives. The issues of specificity and sensitivity must be determined. Environmental detectors must be able to detect low concentrations of threat organisms in a large background of similar non-threat organisms. Finally, environmental samples are highly variable in composition and can contain substances that can interfere with assays. A significant advantage to microarrays is that they allow the inclusion of a number of targets (multiplex detection) as well as proper controls that alert users of potential problems in the samples.
We are also studying the stability and sensitivity of the fluorescent reporter molecules (i.e. the proteins and dyes) used in these assays. We are also studying their application in a variety of different platforms - including suspension arrays, flow cytometer measurements, and microscopy.
2. Characterization of Biological Reference Materials for the Testing of Methods for the Collection, Detection, and Remediation of Biothreat Agents
We are working with the Department of Homeland Security (DHS) and other government agencies to determine the best manner that NIST can meet the needs for data, measurements, and reference materials for the detection of biothreat agents. One area that has been identified is the matter of charactering reference materials that can safely be used for the testing of detection devices, remediation technologies, and personnel training. Lack of uniform and well-characterized testing materials makes it impossible to compare different technologies. We are developing potential reference materials and surrogates. We will characterize these materials and determine the stability as standards. We will use these well-characterized surrogates as model systems to study and improve the instruments and devices used for the detection of these biological threats. The methods for characterization, we are using include classical microbiological techniques, flow cytometry, May 9, 2008up a biological safety level 2 laboratory that will allow us to develop reference materials based on Bacillus anthracis (Sterne vaccine strain) and ricin.
3. Methods for Water Safety in Buildings
We have recently started a collaborative project with the Building and Fire Research Laboratory (BFRL) to improve the safety of building water systems. We are studying the fate and disinfection of biological threats in building water systems. The goals of this project are to provide measurements and guidance that will assist in the remediation of building water systems if exposed to biological threats. We will do measurements on several scales including bench and large-scale (building) levels. This project will measure adhesion of bacteria and toxins to water systems. We are developing simulants that will allow us to do the large-scale experiments in a safe manner. We will compare the results obtained with the simulants to the real threats using small-scale experiments under biosafety level 2 conditions. We are growing biofilms on different plumbing materials using commercial biofilm reactors. The adhesion of the threats and their removal or inactivation will be measured. We will make the measurements using plumbing materials and conditions found in real building water systems. Methods to disinfect (remove and inactivate) the biological threats from building water systems will also be studied.
Expertise
- Immunoassays
- Molecular biology techniques
- Enzyme assays
- DNA extraction and measurement
- Electrophoresis
- Real-time PCR detection
- Bacterial quantitation
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