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Microarrays Meet Point of Care Diagnostics Scott Goebel, Research Scientist, Emerging Technologies Laboratory; Applied Genomic Technologies Core Wadsworth Center, New York State Department of Health Point of Care Diagnostics will require inexpensive, sensitive, multiplexed, rapid assays with walk away hardware and automated results determination so nonscientific operators can understand the results of diagnosis. Low density hydrogel microarrays offer just such qualities and are being developed though collaboration of industry with public health laboratories. Several panels of tests are being developed for respiratory diseases, tuberculosis, and bioterrorist agents. Regulatory hurdles are being addressed during the development phase of these assays to expedite clinical approval. 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. 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. Department of Homeland Security’s Lessons Learned Information Sharing Network Leiloni Stainsby, Manager, Outreach and Partnerships, Lessons Learned Information Sharing, a Nationwide Program of the US Department of Homeland Security Lessons Learned Information Sharing (LLIS.gov) is the national, online network of lessons learned, best practices, and innovative ideas for the emergency response and homeland security communities. LLIS.gov helps to improve homeland security and emergency preparedness nationwide by providing Federal, State, and local responders with a wealth of information and front-line expertise on effective planning, training, and operational practices across homeland security functional areas. Sponsored by the Department of Homeland Security’s Federal Emergency Management Agency, LLIS.gov helps emergency response providers and homeland security officials prevent, protect against, respond to, and recover from terrorist attacks, major disasters, and other emergencies. For more information and to register, visit www.LLIS.gov. 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. Systems Approach to Detection Assays: Achieving High Throughput and High Content P. Scott White, PhD, Team Leader, Biosecurity Applications and Analysis, Bioscience Division, Los Alamos National Laboratory* We have developed multiplexing strategies for a wide variety of applications that address key bottlenecks of existing approaches. The method uses a novel assay that combines fragment detection with genotyping that reverses the “PCR first” paradigm by performing detection and genotyping steps prior to signal amplification and detection. We will show results using mixed marker multiplexed assays that detect bacterial pathogens and characterize them for antibiotic resistance. This approach can be easily adapted to genetic screening or genotyping applications, among others. *In collaboration with: A.Deshpande, J.Gans, Y.Kunde, Po-E Li, J.Song, M.Vuyisich, and M.Wolinsky 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 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 60 minutes), allows real time quantitation of influenza viral neuraminidase activity, is highly sensitive, uses simple operation procedure, and is suitable for diagnosis and large scale antiviral drug resistance surveillance. Molecular Target Discovery for Application in Nucleic Acid Based Diagnostics for Pathogens. Thomas Barry, PhD, R&D Principal Investigator, Molecular Diagnostics Research Group (MDRG), National Centre for Biomedical Engineering Science, National University of Ireland, Ireland We have developed a suite of platform technologies, based on proprietary nucleic acid sequence targets, for the detection and identification of bacteria and fungi. These target technologies can be applied in a wide variety of nucleic acid-based test formats. They have been commercialised through licensing agreements and co-development collaborations with major international diagnostics companies. Currently, the group is working on a 4 year collaborative R+D project with Beckman Coulter Inc. to develop molecular diagnostics for clinically relevant bacterial and fungal pathogens. Rapid Nucleic Acid Diagnostics for the Detection of Microorganisms Andrew P. Miller, PhD, CEO, Ionian Technologies, Inc. Ionian’s rapid isothermal DNA and RNA amplification and detection technologies allow for unparalleled detection of bacterial and viral pathogens in 5-10 minutes. The sensitivity and specificity of DNA/RNA amplification provide significantly improved performance relative to immunoassays, allowing for detection of less than 50 genome equivalents. Results can be read-out with a variety of methods, including fluorescent and lateral flow approaches. Our detection technology combined with a simple heating/detection unit provides nucleic acid testing in a simple point-of-care format. In addition to the POC opportunities, Ionian is also commercializing assays for biothreat detection, agriculture, food safety, and environmental applications. 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. Novel Labeless, Immunosensor Platform Technology Paul A. Millner, PhD, Biosensors and Biocatalysis Group Leader, IMSB, Faculty of Biological Sciences University of Leeds, United Kingdom* Affinity based biosensors are of significant interest since the range of potential targets is virtually limitless and the EU project ELISHA has been successful in demonstrating labeless, electrochemical immunosensor detection of over 15 different target analytes, including viruses. The ELISHA platform can be thought of as an enabling technology, where inclusion of any affinity reagent confers the specificity of that reagent. Antibodies have been demonstrated in the main, however other binding proteins and calixarenes have also been shown to give specific responses. The ELISHA platform is being commercialized by a new spin-out company, ELISHA Systems Ltd (ESL). *In collaboration with: Tim Gibson, ELISHA Systems Ltd. Faster Than Fast: A 1D Solid Phase Capture Immuno Array Format Tom A. Beumer, PhD, Senior Staff Scientist, bioMerieux BV, Netherlands A measurement concept for a solid phase capture (immuno) assay array was developed that is based on a droplet of limited size containing sample and label that periodically moves over a fixed trajectory of considerably larger dimensions. On this trajectory one or more specific capture zones can be defined, on which the particle label is captured in case the molecule of interest is present in the reaction mix. The more often the droplet passes the capture zone, the more particles are captured, thus enabling real time visual or instrumental detection. Reagents, like Eg the label and buffer components - can be stored in the same device. This technology makes optimum use of available molecules and virtually removes diffusion limitation in the binding process, making the concept very fast. Enhanced Europium Nanoparticle-Based Immunoassay for Sensitive Detection of Anthrax Toxin Shixing Tang, MD, PhD, Laboratory of Molecular Virology, DETTD/OBRR/CBER, Food and Drug Administration* A europium (Eu+ ) nanoparticle (NP)-based immunoassay (ENIA) has been developed and exhibited approximately 100-fold more sensitive than ELISA in detecting anthrax toxin. No false positive results were observed in normal serum samples, mouse plasma without PA, or plasma samples collected from mice injected with anthrax lethal factor (LF) or edema factor (EF) alone. For detection of plasma samples spiked with PA, the detection sensitivity for ENIA and ELISA was 100% (11/11) and 36.4% (4/11), respectively. Anthrax toxin can be detected in toxin injected or B. antharacis infected mice plasma samples. These results indicate that the universal labeling technology based on Eu+ NP and its application may provide a rapid and sensitive testing platform for clinical diagnosis and laboratory research. *In collaboration with: J.Zhao, I.K.Hewlett, FDA; M.Moayeri, S.H.Leppla, Z.Chen, R.H.Purcell, NIAID/NIH; Harri Harma, University of Turku, Finland Rapid and Specific CBWA Detection and Identification: Potentiometric Sensing and Monitoring of Bacterial Spore - Strain Recognition Janet Y. Zhou, PhD, and Kalle Levon, PhD, Dept of Chemical and Biological Sciences, Polytechnic University* Potentiometry is the field of electroanalytical chemistry in which potential is measured under the conditions of no current flow. The outer face of macromolecular biological assemblies such as viruses or bacteria carries charged or chargeable groups, such as glycoproteins or lipids. The recognition elements for Bacillus spores, such as peptides, lectins, which have been shown to bind selectively to different Bacillus spores, were immobilized to the electrode surface by polysiloxane film immobilization (PFI). Therefore, the charge compensation and hydrophobic interaction between ligands and Bacillus spores could be observed and monitored by potentiometry. The recognition mechanism has been thoroughly studied, and corresponding potentiometric sensors were developed. The resulting portable sensor device could be used in real-time to detect bacterial spores with very low limit of detection (a few spore per 100 ml solution) without any labeling, and also differentiate the same bacterial spores with different strain. *In collaboration with: C.L.Turnbough, U. Alabama at Birmingham.