Biodetection Technologies: Technological Responses to Biological Threats - 6th Edition
Knowledge Press, February 2010, Pages: 330
The 6th edition in our Detection Technology series is an internationally recognized publication for experts in detection & identification of biological threats.
This publication will explore the latest R&D developments including:
- Point-of-care/clinical applications for pathogen/virus/threat detection and identification
- Technological challenges for rapid/early/specific sensitive detection
- Role of nanotechnology and system miniaturization
- Bioinformatics for biodefense
- Reducing false positives vs. detector sensitivity
- Field-deployable devices: portability/compatibility/reliability/scalability
- Reagentless vs. reagent biodetection systems
- Non-PCR vs. PCR based detection techniques
- Advances in microarray and sequencing technologies
- Issues of sensitivity/testing/evaluation/validation
CHAPTER 1 - Multiplex Real-Time PCR Assay Design for Pathogen Detection, Quantification and Speciation
Robert S. Tebbs, PhD, Staff Scientist, Applied Biosystems*
A multiplex real-time PCR assay was designed to identify 3 species of Vibrio and an internal positive control using a four-dye configuration. Multiple strains were sequenced to identify target sites. Several individual assays within the multiplex contain more than one primer or probe due to strain to strain polymorphisms. Vibrio cholerae, V. parahaemolyticus, and V. vulnificus were detected either individually or in mixtures at =25 genomic copies. The Vibrio multiplex assay showed 100% specificity to all targets analyzed and no detection of an exclusion panel that included nearest neighbors. Each assay in the multiplex exhibited 100 ± 10% efficiency over a 5 log range. Multiplex real-time PCR can simplify pathogen detection and reduce costs since 3 species can be analyzed in a single reaction tube. *In collaboration with: P.M.Brzoska, S.Bit, M.R.Furtado, and O.V.Petrauskene
CHAPTER 2 - Multiplexed Diagnostic Assays for Viral Pathogens of Cattle and Swine
Max V. Rasmussen, PhD, Biomedical Scientist, Biosciences Defense Division, Lawrence Livermore National Laboratory
Diagnostic tests for animal pathogens support a healthy livestock industry and reliable food supply. Lawrence Livermore National Laboratory developed multiplexed assays that simultaneously discriminate nucleic acid sequences of foreign and endemic viral pathogens affecting cattle and swine in a high-throughput format. The porcine-disease assay detects foot-and-mouth disease, vesicular stomatitis, swine vesicular disease, vesicular exanthema of swine and porcine reproductive and respiratory syndrome. The bovine-disease assay detects foot-and-mouth disease, malignant catarrhal fever, rinderpest, bovine viral diarrhea, bluetongue, bovine papular stomatitis, psuedocowpox, infectious bovine rhinotracheitis, vesicular stomatitis and bovine herpes mammalitis. Ongoing DHS-funded research includes enhancing sensitivity to FMDV, supporting the USDA-APHIS assay validation process and development of a FMDV sub-typing microarray.
CHAPTER 3 - Challenges for Sample Preparation and Pathogen Detection: Perspectives from the Food Industry
Daniel R. DeMarco, PhD, Senior Research Microbiologist, DuPont Qualicon
Many promising new sensor technologies purport to have potential applications in foodborne pathogen detection. Often there is little real understanding of the complexities and issues involved. In contrast to air or clinical samples which are relatively simple, food matrix presents a host of unique challenges that must be overcome before adoption of any particular sensing technology will occur in the food industry. These include but are not limited to; sensitivity requirements, sample size issues, matrix diversity, and presence of assay specific inhibitors. In addition, the food industry is notoriously low cost and often low tech in its historically preferred approaches to pathogen detection. This talk will highlight specific examples of the challenges faced for pathogen detection in foods, discuss some history and background of past and currently in-use technologies, and discuss future trends.
CHAPTER 4 - Rapid and On-Demand Detection and Characterization of Staphylococci Causing Bloodstream Infections
Yi-Wei Tang, PhD, Associate Professor of Pathology and Medicine, Director, Molecular Infectious Diseases Laboratory, Vanderbilt University Medical Center
Phenotypic methods take several days for identification and antimicrobial susceptibility testing of staphylococcal isolates after gram-positive cocci in clusters (GPCC) are observed in positive blood cultures. We developed and validated a StaphPlex system for species-level identification of staphylococci, detection of genes encoding Panton-Valentine leukocidin (PVL), and antimicrobial resistance determinants of staphylococci. The StaphPlex system was compared to phenotypic methods for organism identification and antimicrobial resistance detection for positive blood culture specimens in which GPCC were observed. Among a total of 360 GPCC specimens, 273 (75.8%), 37 (10.3%), 37 (10.3%), 1 (0.3%), 3 (0.8%), and 9 (2.5%) were identified by StaphPlex as coagulase-negative Staphylococcus (CoNS), methicillin-resistant Staphylococcus aureus (MRSA), methicillin-susceptible S. aureus (MSSA), or mixed infections of CoNS and MRSA, CoNS and MSSA, or nonstaphylococci, respectively, with an overall accuracy of 91.7%. The 277 CoNS-containing specimens were further identified to the species level with an overall accuracy of 80.1% compared to a combined reference identification. High very major errors were noticed when detection of aacA, ermA, ermC, tetM, and tetK was used to predict in vitro antimicrobial resistance, but relatively few major errors were observed when the absence of these genes was used to predict susceptibility. The StaphPlex system demonstrated high sensitivity and specificity when used for staphylococcal cassette chromosome mec typing and PVL detection. StaphPlex provides simultaneous staphylococcal identification and detection of PVL and antimicrobial resistance determinants within 5 h, significantly shortening the time needed for phenotypic identification and antimicrobial susceptibility testing.
CHAPTER 5 - Immunochemical Technologies for Replacement of Rodent Bioassays in Sensitive Detection of Toxins in Foods
John Mark Carter, PhD, Research Leader, Foodborne Contaminants Research Unit, Agricultural Research Service, US Department of Agriculture*
Rapid sensitive assays for biothreat toxins that can be used to detect intentionally contaminated foods are now typically performed via bioassay in live mice. While bioassay provides essential data on bioavailability, animal models are technically, fiscally, and ethically challenging. Through careful application of state-of-the-art techniques for immunization and screening, we created new monoclonal antibody reagents (MAb) specific for detection of botulinum neurotoxin (BoNT). These MAbs bind BoNT so tightly that, in a sandwich ELISA, they are more sensitive than the rodent bioassay. These reagents are also useful for sample preparation and production of portable tests for field use. Through a CRADA with Safeguard Biosystems Corp., we used these MAbs to develop a simple “dipstick” assay that can detect BoNT in food at levels well below the human oral LD50. We also used the new MAbs to develop sample preparation methods based on immunomagnetic beads. In liquefied food extracts these beads rapidly and irreversibly bind all toxin present in a large sample. Sequestering the beads with a magnet effectively concentrates the toxin into a small volume suitable for laboratory testing. While the toxin is still bound to the beads, we can detect its highly specific peptidase activity using a fluorescence (FRET) based substrate, for detection of sublethal amounts of BoNT in foods.
* In collaboration with: L.Cheng, X.He, R.Rasooly, L.Stanker, and D.Brandon, USDA
CHAPTER 6 - SCODA Electrophoresis for Biomolecule Concentration
Andre Marziali, PhD, President and CSO, Boreal Genomics Inc., Canada
Boreal Genomics has developed a high performance instrument based on SCODA, a novel electrophoretic concentration technology, for efficiently purifying and concentrating biomolecules. Boreal’s technology offers unique advantages in biomolecule concentration including exceptional rejection of PCR inhibitors, an unparalleled ability to enrich for low abundance nucleic acids, and ability to length-select nucleic fragments. We present recent advances in the SCODA technology, including reduction of sample processing times to < 10 min., and applications to protein concentration.
CHAPTER 7 - High Efficiency Bio-Agent Recovery from Simulated Environmental Surfaces with Robust Liquid Rinse Vacuum Collection System
Jared G. Maughan, Vice President of Microbiology & Operations, Microbial-Vac Systems, Inc.
Personnel responsible for sampling must have the capabilities to confidently and safely collect samples that are accurately indicative of the surface contents within suspect contaminated zones. Currently utilized swipe/swab sampling methods are highly restricted in their abilities to effectively cover sufficient surface areas needed to accurately project contaminated area boundaries. The Microbial-Vac System is a novel wet-vacuum pathogen collection system designed to sample large surface areas with increased extraction efficiency. In this study, common environmental surfaces were inoculated and sampled with the M-Vac and swipe/swab methods. The M-Vac demonstrated significantly higher surface bacteria extraction than conventional sampling devices. *In collaboration with: K.J.Church, B.J. Bradley
CHAPTER 8 - Simple, Rapid Visual Indicator of Microbial Contamination
Stephanie M. Martin, PhD, Senior Research Scientist, Kimberly-Clark Corporation
Kimberly-Clark Corporation has identified a dye with a unique ability to rapidly decolorize upon exposure to both gram-positive and gram-negative bacteria, exhibiting a change from blue to colorless in seconds to minutes. The dye represents an extremely versatile detection platform, as it can be applied onto numerous substrates or used in solution as an indicator spray. It may also offer clinical benefit as a diagnostic aid by rapidly distinguishing between bacterial and viral infections. This talk aims to describe this new to the world technology in greater detail.
CHAPTER 9 - Nanoparticles and Quantum Dots for Biodiagnostic Applications
Arnold Kell, PhD, and Benoit Simard, PhD, Principal Research Officer, Steacie Institute for Molecular Sciences, National Research Council, Canada*
Infectious and chronic diseases and bioterrorism are of tremendous global concern. The National Research Council of Canada is dedicated to developing nanoparticle platforms to aid in the rapid detection of these deadly diseases and security threats. Specifically, this presentation will highlight our efforts to utilize unique surface ligands anchored to magnetic nanoparticles, dye-doped silica nanoparticles and quantum dots to enable the selective labeling, magnetic confinement and optical detection of a variety of potentially harmful bacteria and cells in biological samples.
*In collaboration with: Kui Yu
CHAPTER 10 - Point-Of-Care Detection of Staphylococcal Enterotoxins
Hugh Bruck, PhD, Associate Professor of Mechanical Engineering, University of Maryland
Staphylococcal enterotoxins (SEs) are amajor cause of food-borne diseases. Traditionally, SEs assayed immunologically with ELISA. Carbon nanotubes’ (CNT) uniquemechanical and electronic properties combined with a large specific surface areamake them attractive for biosensing.We applied CNT to increase the sensitivity of a simple and portable point-of-care immunosensor based on the detection of Enhanced chemiluminescence (ECL) by a cooled Charge- Coupled Device (CCD) detector. This combination of ECL, CNT and CCD technologies is used to improve the detection of Staphylococcal Enterotoxin B (SEB) in food. Anti-SEB primary antibodies were immobilized onto the CNT surface and the antibody-nanotube mixture was immobilized onto a polycarbonate surface. SEB was then detected by a sandwich-type ELISA assay on the CNTpolycarbonate surface with an ECL assay. SEB in buffer, soymilk, apple juice, andmeat baby food was assayed with a limit of SEB with CCD detector is 0.01 ng/mL similar to the detection limit of a fluorometric detector, which is farmore sensitive than the conventional ELISA detection limit of ~1 ng/ml. Our simple, versatile and inexpensive CCD based Point-of-care detector combined with the CNT-ECL immunosensormethod can be used to simplify and increase sensitivity formany other types of diagnostics and detection assays.
CHAPTER 11 - Low Cost, LED-Based xMAP Analyzer for Multiplexed Diagnosis and Environmental Detection of Biological Agents
Amy L. Altman, PhD, Director, Extramural Research Office, Luminex Corporation
Open-architecture xMAP® technology, developed by Luminex, is ideally suited to a wide range of applications throughout the detection industry. xMAP technology allows simultaneous detection of bacterial, viral and toxin agents, in a highly flexible, multiplexed architecture. Recently, improvements built upon the core Luminex analyzer technologies have resulted in the development of MagPix; a low-cost, compact, rugged, diagnostic and environmental testing xMAP analyzer. This instrument moves away from a flow cytometry-based system to an instrument that employs Light Emitting Diodes (LEDs) and a CCD imager, coupled with an improved magnetic microsphere-based array (MagPlex™).
CHAPTER 12 - Intelligent Wound Management: In-situ Sensors to Detect Infection
Duncan Sharp, PhD, Research Associate, Nottingham Trent University, United Kingdom
The development of a prototype sensor suitable for assessing wound integrity to alert the patient or clinicians to the onset of bacterial colonization by key organisms is described. Based on a multilayer laminate fabrication process, mass production of identical yet inherently disposable sensors suitable for direct incorporation within conventional wound dressings can be readily achieved. The design characteristics and application to the intelligent management of problematic burn wounds are described.
CHAPTER 13 - Development of the BioSeeq-Clinical System, Providing “Sample in, answer out” PCR Testing Capability for Point-of-Care Diagnosis
John W. Czajka, PhD, MBA, Director Technology Development, Smiths Detection Diagnostics
Smiths Detection Diagnostics is currently developing the BioSeeq-Clinical System for the identification of hospital associated infections. The system utilizes a single-use sample preparation consumable that is driven by the BioSeeq instrument, providing true “Sample in, answer out” capability. A highly multiplexed LATE-PCR assay for MRSA is currently being developed and evaluated for use with the BioSeeq-Clinical System for pre-admission screening in hospitals.
CHAPTER 14 - Taking Biological Detectors to the Sample: A Paradigm Shift for Sample Preparation
Charles C. Young, PhD, Senior Staff Scientist, The Johns Hopkins University Applied Physics Laboratory
Next generation biosensors are envisioned to be small enough to carry into the field and generate near real time results using multiple sensing modalities to yield orthogonal confirmation for the presence of biological agents in difficult matrices without the requirement for culture. These sensors must integrate sample preparation, agent detection, and data fusion/analysis algorithms in a single footprint so that system operation can be performed by relatively untrained users. For these future systems sample preparation will remain a critical challenge, requiring co-purification of nucleic acids, proteins, lipids, and small molecules in a single, universal approach. Toward this goal, we have developed a “proof of concept” electrophoretic format based on isotachophoresis that simultaneously purifies and concentrates nucleic acids and proteins from humic acids and food matrices. At the present time, greater than 40-fold concentrations of both protein and DNA have been achieved and even greater concentration of 400-fold or greater should be possible in the proposed test bed and breadboard systems. Additionally, complete separation of proteins/DNA from humic acids and food matrices has resulted in 100-1000 fold improvements in PCR amplification efficiencies. Initial studies on the use of the method for small molecule and RNA purification are just beginning, but we believe that isotachophoresis represents one of the few solutions for a truly universal sample preparation approach in which all diagnostically important targets can be concentrated, isolated, concentrated and purified.
CHAPTER 15 - Bacteriophage Based Signal Amplification: The Solution to Noise Pollution
Lawrence D. Goodridge, PhD, Assistant Professor of Food Microbiology, Dept of Animal Sciences, Colorado State University
Signal amplification may be defined as the use of specific detection methodologies to directly increase the signal to noise ratio in proportion to the amount of target in the reaction. Here, we take advantage of the fact that phages produce multiple progeny during infection to develop a rapid and sensitive assay for detection of bacterial pathogens. We also demonstrate the use of phage amplification to develop an assay in which the phages themselves are the target organism, as indicators of biological water quality.
CHAPTER 16 - Five Questions to Answer before Implementing Biological Field Testing
Kathryn Hansen, Bioterriorism Response, Microbial Diseases Lab, California Department of Public Health
Following the Anthrax Letters, a demand for field identification of biological agents led to a plethora of commercially developed assays to fulfill this perceived need. This presentation will outline questions participants should ask before deciding to implement field tests to detect or identify biological agents. Using the tools presented, participants can develop answers enabling them to determine if a field test is likely to be reliable and appropriately useful, justify costly investments, integrate field decisions concerning when and when not to do field testing, coordinate field testing with outside agency requirements, and provide a sound basis for interpreting results.
CHAPTER 17 - Early Warning of Microbiological Contamination of Water
Yuliya Shakalisava, PhD, Research Fellow, Chemical Sciences - Adaptive Sensors Group, Dublin City University, Ireland
Microbial contamination of public water resources represents serious health risk. Monitoring of microbial water quality rely on culture growth methods and require at least 18-24 hours for analysis. This is not acceptable in cases where immediate action has to be taken. The presented work evaluates several strategies for utilizing surrogate measurements of water quality, such as turbidity, particle size and video sensing, as early warning systems for microbiological contamination, using parallel cell-based measurements for reference measurements. Results from initial field studies will be presented, along with a discussion on potential strategies for maximizing the usefulness of the surrogate approaches, in particular, through minimization of false positives and false negatives.
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