Biodetection Technologies: Technological Responses To Biological Threats - 5th Edition

With an emphasis on rapid detection and low-cost identification, this publication provides solutions to the challenges presented by the evolving pattern of complex biological threat agents of various origins.

Completely updated with the latest information, including narratives, charts, graphs and question & answers, this essential reference tool addresses the following important topics:

- PCR and Non-PCR Based Detection
- Reagent and Reagentless Based Detection
- Point-of-Care Toxin & Pathogen Detection
- Utilizing Bacteriophage for Biological Threat Response
- Real-World Sample Preparation for Toxin & Pathogen Detection

Chapter 1 - The Neccesity for Improving Biodetection Technologies
Robert Hooks, Deputy Assistant Secretary for WMD and Biodefense, U.S. Department of Homeland Security
In the biodefense area, DHS efforts focus on providing leadership by coordinating with other Federal partners, state/local/tribal jurisdictions, the private sector and the international community. In addition to developing technologies for Biowatch or NBIS, which are two DHS program areas, improved technology is equally important for our partners and is critical to improving the nation’s capabilities in the biodefense arena.

Chapter 2 - SAFE: Sequencing for Avian Flu Epidemic
Niveen Mulholland, PhD, Senior Scientist, MRI-NCR, Midwest Research Institute*
We have developed a system for detecting mutations that would give rise to potential pandemic-causing influenza strains. The system, SAFE: Sequencing for Avian Flu Epidemic, first uses real time RT-PCR to detect H5, the highly pathogenic avian influenza most likely to cause a pandemic. We next use pyrosequencing to detect codon changes encoding amino acids known to define human versus avian influenza signatures. The SAFE real time RT-PCR assay specifically detects H5 in a multiplex reaction designed to detect a region of the Matrix gene common to all Influenza A subtypes. The H5 primer/probe set is specific; it does not cross react with other Influenza A subtypes tested. The M primer/probe set serves as an internal control, detecting all subtypes tested. Pyrosequencing assays were developed to screen, at the nucleotide level, for 52 amino acids changes defined by Chen et al (2006) to be avian- or human- specific. A library has been built to screen the sequence data generated and properly identify the strain in question as a potential threat. This surveillance system described here will allow the global community to monitor for H5N1 and for mutations that will render the virus more infective and virulent to humans. *In collaboration with: N.Waybright, E.Petrangelo, P.Lowary & J.Bogan

Chapter 3 - Next Generation Automated Multi-Target Detection Platform for Closer to Source Diagnostics
Todd Ritter, CEO of Applied Science & Technology, Idaho Technology Inc.
Idaho Technology has developed a highly multiplexed detection system capable of concurrently identifying and genetically discriminating dozens of viruses and bacteria. The syringe-loaded system utilizes a flexible plastic pouch combining automated sample preparation, reverse transcription for RNA viruses, and two-stage nested multiplex PCR performing 120 discreet analyses simultaneously. Capable of processing a variety of sample types, the small, lightweight diagnostic system represents a next generation in automated detection systems.

Chapter 4 - Rapid Multiplexed Nucleic Acid and Antibody Based Sensor for Biothreat Detection
Michael R. Meyer, Director of Laboratories, ICx Biosystems
RapidPlex is a fully automated, triggered confirmation system designed for detection and identification of bacteria, virus, and toxin threats in 10 minutes. The RapidPlex system provides simultaneous, multiplexed detection of protein and DNA/RNA markers through parallel antibody and nucleic acid-based assays. Antibody-based detection utilizes multiplexed sandwich assays on spectrally encoded microspheres followed by high resolution imaging of individual beads for detection of spores/cells captured on the bead surfaces as well as detection of toxins and viruses coating the bead surfaces. The nucleic acid-based process includes automated cell lysis and purification, followed by multiplexed DNA/RNA amplification and detection of the DNA amplicons on spectrally encoded microspheres.

Chapter 5 - Multiplex Detection of Biothreat Agents by Fluidic Force Discrimination
Gary W. Long, PhD, Vice President and Senior Scientist, Tetracore, Inc.
Fluidic Force discrimination (FFD) has recently been applied to the detection of Biothreat agents. In this talk, we will describe the multiplex detection of 3 or more agents by an immunoassay developed for FFD, and demonstrate that sensitivity of detection is similar or greater than that obtained in lateral flow devices and by ELISA. We will also describe improvements to the sensitivity of detection of nucleic acids using this technology.

Chapter 6 - Use of CANARY™ for Rapid, Automated Collection and Analysis of Bioaerosol Samples
Thomas Hazel, PhD, Vice President Research, Innovative Biosensors, Inc.
CANARY™ is a cell-based technology that enables rapid identification of bacterial, viral, and toxin targets in liquid or aerosol samples. In this presentation we describe the testing and validation of our new BioFlash™ instrumentation, designed to provide ‘detect-to-protect’ capability for environmental monitoring and building security applications. This platform enables automated collection, detection, and simultaneous identification of up to 21 target aerosol agents with increased speed and sensitivity.

Chapter 7 - SAW RFID Biosensors for Ubiquitous Molecular Recognition
William D. Hunt, PhD, Professor, School of Electrical and Computer Engineering, Georgia Institute of Technology*
Research by Hunt’s group at Georgia Tech has demonstrated that an acoustic wave biosensor with a biolayer coating can detect analytes such as Bascillus spores, cocaine, TNT/RDX in the gaseous phase. A new embodiment of this approach involves a SAW RFID/biosensor, which can passively sense and report on molecules in its environment. The SAW RFID/biosensor system involves a main interrogation unit transmitting an RF signal to a passive SAW structure located a short distance away. The device combined with the antenna re-transmits a reflected acoustic wave, which has been perturbed by the on-chip molecular recognition events. *In collaboration with: P.J.Edmonson, D.D.Stubbs, Zen Sensing LLC; D.Bertieri, Penn United Technologies Inc.

Chapter 8 - TIRF-EC Biosensors with Reagentless Bioassays for Rapid and Accurate Detection of Pathogens
Alexander N. Asanov, PhD, President & Scientific Director, TIRF Technologies
This presentation will describe revolutionary new portable and handheld biosensors based on Total Internal Reflection Fluorescence (TIRF) combined with ElectroChemistry (TIRF-EC). TIRF-EC sensors are capable of detecting hundreds of DNA/RNA and protein signatures of multiple pathogens in a matter of several seconds. The detection limit is at the level of single molecules. In conjunction with reagentless assays TIRF-EC sensors require no or minimum sample preparation stages. The sensors are suitable for a variety of biomedical point-of-care and biodefense field applications.

Chapter 9 - Cost-Efficient Technological Solutions for Warning-Type Bioaerosol Detection
Victor N. Morozov, PhD, Research Professor, Manager of a Laboratory of Detection & Diagnostics, George Mason University, The National Center for Biodefense and Infectious Diseases (NCBID)
Rapid ultra-sensitive assay of aerosolized pathogens presents a tremendous challenge in biodetection. NCBID has developed two key technologies solving major ‘bottle-necks’ in such detection: (i) energy-consuming collection of aerosol and (ii) slow, diffusion-controlled immunoassay. We demonstrated that collection of aerosol on water-soluble nano-filters combined with electrophoresis-assisted immunoassay and active detection of collected pathogens with magnetic beads enable detection of zeptomolar amounts of pathogens (150-500 molecules or viruses) in 2-3 minutes.

Chapter 10 - Bridging Between the Worlds: When ‘Proteomics’, ‘Ionics’, and ‘Electronics’ Meet
Ilan Levy, PhD, Bioelectronic Chip Research Project, Intel Research Israel, Corporate Technology Group, Intel Corporation, Israel
Over the years, significant inflection points in understanding and delivering health care advances have occurred, particularly with the convergence of technologies. Today’s ecosystem of aging populations, increased number of chronic patients, a global shortage of, physicians, nurses and hospital beds, enforces major market shift towards home health care. While some initial technologies for remote patient monitoring are already in trials, fuller and more elaborated solutions are sought for. Cost effective medical diagnostics techniques, can be executed at point of care (PoC), deliver immediate results, and be connected to the rest of the health care information systems. It will enable quality health care services that will not only overcome the shortages described above, but will also enable more precise and personalized medicine. The unique technologies developed at Intel have the potential of redefining modern health care, thus enabling the broad range implementation of personalized medicine and PoC practice.

Chapter 11 - Activity Detector for Botulinum Neurotoxins (BoNT)
Avi Rasooly, PhD, Center for Devices and Radiological Health, Office of Science and Engineering Laboratories, Division of Biological Sciences, U.S. Food & Drug Administration and the National Cancer Institute, Cancer Diagnosis Program
We developed an activity detector for Botulinum Neurotoxins (BoNT) based on cleavage of a specific target peptide (SNARE peptide such as SNAP-25) as an alternative to the existing FDA mouse bioassay. We tested this assay with a portable microfluidics chip and a CCD based detector developed to carry out BoNT activity assays. The detector system consists of two modules; (1) the detection module which houses the CCD camera and emission filters and (2) the excitation and sample module, containing the illumination source, the excitation filter and the 9-well sample chip. The FRET activity assay used in this study utilized a FITC/DABCYL-SNAP-25 peptide substrate in which cleavage of the substrate by BoTN/A produced an increase in fluorescence emission, measured using the EL-CCD detector platform. Limits of detection (LODs) were between 0.625-1.25 nM (31-62 ng/ml) for LcA and 0.313 nM (45 ng/ml) for the full toxin, BoTN/A, results that are similar to standard fluorescent plate reader measurements.

Chapter 12 - Protein Based Assays for Novel Viral Detection and Serologic Assays for Detection of Poxviruses Including Smallpox Virus; Applications for Outbreak Response and Surveillance
Kevin L. Karem, PhD, Microbiologist, National Center for Zoonotic, Vector-Borne, and Enteric Diseases (ZVED), Division of Viral and Rickettsial Disease, Centers for Disease Control and Prevention (CDC)
Since the eradication of Smallpox in 1980, concern over the potential use of Smallpox virus (Variola) as an agent of bio-warfare, or terrorism has driven research for new vaccination methods, treatments and detection methods. We have developed and characterized monoclonal antibodies for use in novel viral detection assays based on antigen (protein) recognition, viral protein recognition and insight into detection and recognition of live virus. Detection of virus exposure in the absence of traditional laboratory equipment has also been piloted as a rapid point of care test.

Chapter 13 - A Recombinant Bacteriophage-Based Assay for Discriminative Detection of Culturable and Viable but Nonculturable Escherichia coli O157:H7
Yasunori Tanji, PhD, Associate Professor, Dept of Biotechnology, Tokyo Institute of Technology, Japan
Green fluorescent protein (GFP) labeled bacteriophage, specific to Escherichia coli O157:H7 was used to construct lysozyme- inactivated GFP labeled phage. The new recombinant phage lack lytic activity due to inactivation of gene e, which produces lysozyme responsible for cell lysis. This system allowed the discriminative detection of culturable, viable but nonculturable (VBNC) and dead cells in the stress-induced environment.

Chapter 14 - Landscape Phage-Derived Recognition Interfaces for Detection Devices
Valery A. Petrenko, PhD, DSci, Professor, Dept of Pathobiology, Auburn University
Filamentous phages are thread-shaped bacterial viruses. Their outer coat is a tube formed by thousands equal copies of the major coat protein pVIII. Libraries of random peptides fused to pVIII domains were used for selection of phages probes specific for a panel of test antigens and biological threat agents. Because the viral carrier in the phage borne bio-selective probes is infective, they can be cloned individually and propagated indefinitely without needs of their chemical synthesis or reconstructing. As a new bioselective material, landscape phages combine unique characteristics of affinity reagents and self assembling proteins. Biorecognition layers formed by the phage-derived probes bind biological agents with high affinity and specificity and generate detectable signals in analytical platforms. The performance of phage-derived materials as biorecognition interface was illustrated by detection of Bacillus anthracis spores and Salmonella typhimurium cells. With further refinement, the phage-derived analytical platforms for detecting and monitoring of numerous threat agents may be developed, since phage interface against any bacteria, virus or toxin may be readily selected from the landscape phage libraries. As an interface in the field-use detectors, they may be superior to antibodies, since they are inexpensive, highly specific and strong binders, resistant to high temperatures and environmental stresses.

Chapter 15 - Rapid Label-Free Phage-Based Biosensors for Bacterial Detection
Rosemonde Mandeville, PhD, President and CEO, Biophage Pharma Inc, Canada*
Biophage Pharma Inc. is actively involved in the development and commercialization of environmentally safe solutions for the management of bacterial contamination. Our first commercially available product, the PDS®-16 biosensor can be used for the continuous on-line monitoring of the total bacterial load in biological fluids as well as clinical sensitivity to antibiotics with outstanding reproducibility and sensitivity. The PDS®-16 biosensor can also detect the presence of anthrax spores in a biological sample. This presentation will cover Biophage’s new generation of biosensors using phages as recognition receptors for specific and rapid detection of pathogenic bacteria in field samples.*In collaboration with: M.Zourob, A.Shabani, B.Allain, A.Martineau

Chapter 16 - SEPTIC - SEnsing of Phage-Triggered Ion Cascade
Maria D. King, PhD, Research Scientist, Dept of Mechanical Engineering, Texas A&M University*
In the era of potential bioterrorism and pandemics of antibiotic-resistant microbes, bacterial contamination is a major concern. We have recently developed and demonstrated an entirely new nanoscale technology, called SEPTIC, for SEnsing of Phage-Triggered Ion Cascade. Based on a nanowell chip, SEPTIC has already been shown to be capable of unambiguous identification of live bacteria on a time scale of seconds to minutes. The technology is based on using nanoscale fluctuation analysis to detect the massive ionic fluxes associated with the initial step of bacteriophage infection, the injection of the phage DNA into the cell. *In collaboration with: Laszlo B. Kish, TAMU

Chapter 17 - Biosensing Bacterial Pathogens with Reporter Bacteriophage
Steven A. Ripp, PhD, Research Associate Professor, Center for Environmental Biotechnology, University of Tennessee
When contemplating approaches for the detection of chemical agents, nothing is more diverse, sensitive, robust, manipulatable, or cost-effective than the bacterial cell, and lux-based bioluminescent bioreporters have for over a decade taken acute advantage of this diversity to sense and respond to specific chemical targets. To add biological targets to their sensing repertoire, bioluminescent bioreporters have been linked to bacteriophage/host cell specificity through quorum sensing autoamplification to yield new bioreporter assays capable of detecting distinct bacterial pathogens such as Escherichia coli O157:H7. The integration of these assays with integrated circuit microluminometers, nanofabricated interfaces, and CCD imagers provides both miniaturized lab-on-a-chip and high-throughput real-time monitoring of biological exposures.

Chapter 18 - Sample Collection and Processing: Experiences from the Lab to the Field
Andrew Cannons, PhD, Scientific Director, University of South Florida Center for Biological Defense
Preparation and processing of samples for the detection and characterization of bioterrorism (BT) agents are essential elements for the early recognition of a BT event. This phase includes sample collection in the field to rapid processing of varied matrices in the lab followed by detection and analysis. Here we report on some of our efforts in sample preparation and processing, including sample collection training for emergency responders and processing different real life samples (food, soil, powders) for rapid pathogen characterization.

Chapter 19 - Development of an Automated Sample Preparation Device for Use on the BioSeeq®-Vet Portable PCR System
John W. Czajka, PhD, MBA, Vice President of Technology Acquisition, Smiths Detection
Smiths Detection has developed the BioSeeq®-Vet PCR System, a portable PCR instrument that performs automated sample preparation and PCR analysis. Nucleic acids are extracted and purified from samples by the Sample Preparation Unit (SPU), a single-use, self-contained sample preparation and PCR consumable. All enzymes and reagents needed by the unit are preloaded into the SPU and are temperature stable. The SPU was designed to purify DNA or RNA from a wide range of sample types and is driven by the BioSeeq®-Vet instrument.

Chapter 20 - Advances in Electronic Microarray Technology
Dalibor Hodko, PhD, Director, Advanced Technology, Nanogen, Inc.
An overview of the latest developments and applications of the electronic microarray technology will be given, in particular with respect to on-chip amplification and miniaturization of the platform. The presentation will for the first time demonstrate real-time detection of DNA amplification in an array format. Recent efforts toward integrating sample preparation with the electronic microarray platform will be summarized.

Chapter 21 - A Powerful New Device and Method for Detecting and Concentrating Nucleic Acids from Complex and Dilute Samples
Andre Marziali, PhD, Director, Engineering Physics, Dept of Physics and Astronomy, University of British Columbia
Purification of nucleic acids from complex and dilute samples remains a challenging task for many applications, including pathogen detection, environmental samples, and recovery of high molecular weight DNA. We have developed a novel electrophoretic technology for efficiently purifying and concentrating even very low levels of nucleic acids from samples where the majority of existing isolation techniques fail. We demonstrate direct extraction of DNA from soil and other environmental samples, recovery of high molecular weight DNA, and detection of DNA at zeptomolar concentrations. *In collaboration with: J.Pel, D.Broemeling, R.Attfield, S.Carayon, I.Chan, C.Cowdell, J.Hale, E.Holtham, S.Inglis, K.Lu, G.Mercer, V.Miao, N.Shah, G.Shibahara, L.Whitehead, J.Davies

Chapter 22 - Nested Well Arrays for Toxin Detection
Z. Hugh Fan, PhD, Associate Professor, Interdisciplinary Microsystems Group, Biomedical Engineering, Mechanical & Aerospace Engineering, University of Florida
We have developed a method for detecting toxins that inhibit protein synthesis. Biological synthesis of proteins is implemented in a cell-free medium in an array of nested wells. To detect a toxin, a group of proteins is simultaneously synthesized in the array. The production yields of these proteins are inhibited differentially by the toxin. The toxin can thus be identified based on the unique response pattern of the array.

Chapter 23 - Whole Cell Biosensors: Chip Canaries for Toxicity Detection
Shimshon Belkin, PhD, Professor, Institute of Life Sciences, The Hebrew University of Jerusalem, Israel
Whole cell biosensors, based upon live genetically engineered cells ‘tailored’ to sense toxic compounds, can report on general sample toxicity, avoiding the need to identify specific threat agents. We have pursued several pathways to immobilize such cells onto a solid platform and couple it into a signal transduction apparatus; these include the incorporation of bacterial reporter cells into disposable plastic biochips, and the development of a dedicated toxicity analyzer to analyze their response.

Chapter 24 - Detection of Biological Threat Contaminants in Critical Buildings
Ashok Kumar, PhD, Program Manager, Construction Engineering Research Laboratory, U.S. Army Engineer Research and Development Center
A nanoscale sensing model through Fluorescent Resonant Energy Transfer (FRET) interactions between fluorescent quantum dots (QDs) and organic quencher molecules can be used as a means of multiplexed detection of microbial contaminants in buildings. The detection of antigens can be measured through photoluminescence (PL) in solution. The multiplexing immuno-assay can provide the ability to detect several types of biological threat contaminants simultaneously in near real-time.

Chapter 25 - Fast, Ultrasensitive Virus Detection
Aurel Ymeti, PhD, BioPhysical Engineering, MESA+ Institute for Nanotechnology, University of Twente, The Netherlands
We have developed an ultrasensitive sensor that could potentially be used in a handheld device to, within minutes, detect various viruses and measure their concentration. The sensor could be used to quickly screen people at airports, hospitals and emergency clinics to control outbreaks of diseases such as SARS and the bird flu. All it would take is a tiny sample of saliva, blood, or other body fluid