John M Dzenitis

Lawrence Livermore National Laboratory, Livermore, California, United States

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Publications (19)41.45 Total impact

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    ABSTRACT: Detection of pathogens and relevant genetic markers using their nucleic acid signatures is extremely common due to the inherent specificity genomic sequences provide. One approach for assaying a sample simultaneously for many different targets is the DNA microarray, which consists of several million short nucleic acid sequences (probes) bound to an inexpensive transparent substrate. Typically, complex samples hybridize to the microarray and the pattern of fluorescing probes on the microarray's surface identifies the detected targets. In the case of evolving or newly emergent organisms, a hybridization pattern can occur that differs from any previously known sources. When this happens it can be useful to recover the hybridized DNA from the binding locations of interest for sequencing. Here we present the novel utilization of a focused Infrared (IR) laser to heat user-selected spots on the DNA microarray surface, causing only localized dehybridization and recovery of the desired DNA into an elution buffer where it is available for subsequent amplification or sequencing. The introduction of a focused dehybridization method for spots of interest suppresses the amount of background DNA to be analyzed from downstream processes, and should reduce subsequent sequence assembly errors. This technique could also be applied to high-density protein microarrays where the desire to locally heat spots for release of bound molecules is desired.
    The Analyst 05/2013; · 4.23 Impact Factor
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    ABSTRACT: We developed an extendable open-source Loop-mediated isothermal AMPlification (LAMP) signature design program called LAVA (LAMP Assay Versatile Analysis). LAVA was created in response to limitations of existing LAMP signature programs. LAVA identifies combinations of six primer regions for basic LAMP signatures, or combinations of eight primer regions for LAMP signatures with loop primers, which can be used as LAMP signatures. The identified primers are conserved among target organism sequences. Primer combinations are optimized based on lengths, melting temperatures, and spacing among primer sites. We compare LAMP signature candidates for Staphylococcus aureus created both by LAVA and by PrimerExplorer. We also include signatures from a sample run targeting all strains of Mycobacterium tuberculosis. We have designed and demonstrated new software for identifying signature candidates appropriate for LAMP assays. The software is available for download at http://lava-dna.googlecode.com/.
    BMC Bioinformatics 06/2011; 12:240. · 3.02 Impact Factor
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    ABSTRACT: DNA microarrays contain sequence specific probes arrayed in distinct spots numbering from 10,000 to over 1,000,000, depending on the platform. This tremendous degree of multiplexing gives microarrays great potential for environmental background sampling, broad-spectrum clinical monitoring, and continuous biological threat detection. In practice, their use in these applications is not common due to limited information content, long processing times, and high cost. The work focused on characterizing the phenomena of microarray hybridization and selective release that will allow these limitations to be addressed. This will revolutionize the ways that microarrays can be used for LLNL's Global Security missions. The goals of this project were two-fold: automated faster hybridizations and selective release of hybridized features. The first study area involves hybridization kinetics and mass-transfer effects. the standard hybridization protocol uses an overnight incubation to achieve the best possible signal for any sample type, as well as for convenience in manual processing. There is potential to significantly shorten this time based on better understanding and control of the rate-limiting processes and knowledge of the progress of the hybridization. In the hybridization work, a custom microarray flow cell was used to manipulate the chemical and thermal environment of the array and autonomously image the changes over time during hybridization. The second study area is selective release. Microarrays easily generate hybridization patterns and signatures, but there is still an unmet need for methodologies enabling rapid and selective analysis of these patterns and signatures. Detailed analysis of individual spots by subsequent sequencing could potentially yield significant information for rapidly mutating and emerging (or deliberately engineered) pathogens. In the selective release work, optical energy deposition with coherent light quickly provides the thermal energy to single spots to release hybridized DNA. This work leverages LLNL expertise in optics, microfluids, and bioinformatics.
    01/2011;
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    ABSTRACT: The Environmental Sample Processor (ESP) is a device that allows for the underwater, autonomous application of DNA and protein probe array technologies as a means to remotely identify and quantify, in situ, marine microorganisms and substances they produce. Here, we added functionality to the ESP through the development and incorporation of a module capable of solid-phase nucleic acid extraction and quantitative PCR (qPCR). Samples collected by the instrument were homogenized in a chaotropic buffer compatible with direct detection of ribosomal RNA (rRNA) and nucleic acid purification. From a single sample, both an rRNA community profile and select gene abundances were ascertained. To illustrate this functionality, we focused on bacterioplankton commonly found along the central coast of California and that are known to vary in accordance with different oceanic conditions. DNA probe arrays targeting rRNA revealed the presence of 16S rRNA indicative of marine crenarchaea, SAR11 and marine cyanobacteria; in parallel, qPCR was used to detect 16S rRNA genes from the former two groups and the large subunit RuBisCo gene (rbcL) from Synecchococcus. The PCR-enabled ESP was deployed on a coastal mooring in Monterey Bay for 28 days during the spring-summer upwelling season. The distributions of the targeted bacterioplankon groups were as expected, with the exception of an increase in abundance of marine crenarchaea in anomalous nitrate-rich, low-salinity waters. The unexpected co-occurrence demonstrated the utility of the ESP in detecting novel events relative to previously described distributions of particular bacterioplankton groups. The ESP can easily be configured to detect and enumerate genes and gene products from a wide range of organisms. This study demonstrated for the first time that gene abundances could be assessed autonomously, underwater in near real-time and referenced against prevailing chemical, physical and bulk biological conditions.
    PLoS ONE 01/2011; 6(8):e22522. · 3.53 Impact Factor
  • J M Dzenitis, A J Makarewicz
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    ABSTRACT: We developed, tested, and now operate a civilian biological defense capability that continuously monitors the air for biological threat agents. The Autonomous Pathogen Detection System (APDS) collects, prepares, reads, analyzes, and reports results of multiplexed immunoassays and multiplexed PCR assays using Luminex{copyright} xMAP technology and flow cytometer. The mission we conduct is particularly demanding: continuous monitoring, multiple threat agents, high sensitivity, challenging environments, and ultimately extremely low false positive rates. Here, we introduce the mission requirements and metrics, show the system engineering and analysis framework, and describe the progress to date including early development and current status.
    01/2010;
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    ABSTRACT: We have developed and field-tested a now operational civilian biodefense capability that continuously monitors the air in high-risk locations for biological threat agents. This stand-alone instrument, called the Autonomous Pathogen Detection System (APDS), collects and selectively concentrates particles from the air into liquid samples and analyzes the samples using multiplexed PCR amplification coupled with microsphere array detection. During laboratory testing, we evaluated the APDS instrument's response to Bacillus anthracis and Yersinia pestis by spiking the liquid sample stream with viable spores and cells, bead-beaten lysates, and purified DNA extracts. APDS results were also compared to a manual real-time PCR method. Field data acquired during 74 days of continuous operation at a mass-transit subway station are presented to demonstrate the specificity and reliability of the APDS. The U.S. Department of Homeland Security recently selected the APDS reported herein as the first autonomous detector component of their BioWatch antiterrorism program. This sophisticated field-deployed surveillance capability now generates actionable data in one-tenth the time of manual filter collection and analysis.
    Analytical Chemistry 10/2008; 80(19):7422-9. · 5.82 Impact Factor
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    ABSTRACT: We present a fully automated DNA purification module comprised of a micro-fabricated chip and sequential injection analysis system that is designed for use within autonomous instruments that continuously monitor the environment for the presence of biological threat agents. The chip has an elliptical flow channel containing a bed (3.5 x 3.5 mm) of silica-coated pillars with height, width and center-to-center spacing of 200, 15, and 30 microm, respectively, which provides a relatively large surface area (ca. 3 cm(2)) for DNA capture in the presence of chaotropic agents. We have characterized the effect of various fluidic parameters on extraction performance, including sample input volume, capture flow rate, and elution volume. The flow-through design made the pillar chip completely reusable; carryover was eliminated by flushing lines with sodium hypochlorite and deionized water between assays. A mass balance was conducted to determine the fate of input DNA not recovered in the eluent. The device was capable of purifying and recovering Bacillus anthracis genomic DNA (input masses from 0.32 to 320 pg) from spiked environmental aerosol samples, for subsequent analysis using polymerase chain reaction-based assays.
    The Analyst 03/2008; 133(2):248-55. · 3.97 Impact Factor
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    ABSTRACT: We developed an automated point-of-care diagnostic instrument that is capable of analyzing nasal swab samples for the presence of respiratory diseases. This robust instrument, called FluIDx, performs autonomous multiplexed RT-PCR reactions that are analyzed by microsphere xMAP technology. We evaluated the performance of FluIDx, in comparison rapid tests specific for influenza and respiratory syncytial virus, in a clinical study performed at the UC Davis Medical Center. The clinical study included samples positive for RSV (n = 71), influenza A (n = 16), influenza B (n = 4), adenovirus (n = 5), parainfluenza virus (n = 2), and 44 negative samples, according to a composite reference method. FluIDx and the rapid tests detected 85.9% and 62.0% of the RSV positive samples, respectively. Similar sensitivities were recorded for the influenza B samples; whereas the influenza A samples were poorly detected, likely due to the utilization of an influenza A signature that did not accurately match currently circulating influenza A strains. Data for all pathogens were compiled and indicate that FluIDx is more sensitive than the rapid tests, detecting 74.2% (95% C.I. of 64.7-81.9%) of the positive samples in comparison to 53.6% (95% C.I. of 43.7-63.2%) for the rapid tests. The higher sensitivity of FluIDx was partially offset by a lower specificity, 77.3% versus 100.0%. Overall, these data suggest automated flow-through PCR-based instruments that perform multiplexed assays can successfully screen clinical samples for infectious diseases.
    02/2008;
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    ABSTRACT: A method of detecting real events by obtaining a set of recent signal results, calculating measures of the noise or variation based on the set of recent signal results, calculating an expected baseline value based on the set of recent signal results, determining sample deviation, calculating an allowable deviation by multiplying the sample deviation by a threshold factor, setting an alarm threshold from the baseline value plus or minus the allowable deviation, and determining whether the signal results exceed the alarm threshold.
    01/2008;
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    ABSTRACT: This research investigates the physical collection characteristics of a novel and compact (combined dimensions ) air-to-liquid aerosol collector recently developed by the Lawrence Livermore National Laboratory. In this collector, the air flow is drawn into an annular, centripetal slot, which directs the aerosol flow into a small volume of liquid at the sampler's center, thereby imbedding airborne particles in the liquid. A mist eliminator positioned above the liquid prevents liquid droplets from escaping the sampler thus improving its performance. We found that the sampler's collection efficiency increases with decreasing width of the annular slot, and with increasing flow rate, mist eliminator speed and sample volume. When operating at a flow rate of 275 l/min and collecting particles into 1 ml of liquid, the sampler can achieve concentration rates as high as 275,000/min. We also determined that by changing the sampler's operational parameters we can move its collection efficiency curve over a wide range of particle sizes, thus adjusting the sampler's cutoff size from to. The sampler's small size and its high collection performance make it suitable for many biodetection strategies.
    Journal of Aerosol Science. 01/2006;
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    ABSTRACT: We have developed and tested a fully autonomous pathogen detection system (APDS) capable of continuously monitoring the environment for airborne biological threat agents. The system was developed to provide early warning to civilians in the event of a bioterrorism incident and can be used at high profile events for short-term, intensive monitoring or in major public buildings or transportation nodes for long-term monitoring. The APDS is completely automated, offering continuous aerosol sampling, in-line sample preparation fluidics, multiplexed detection and identification immunoassays, and nucleic acid-based polymerase chain reaction (PCR) amplification and detection. Highly multiplexed antibody-based and duplex nucleic acid-based assays are combined to reduce false positives to a very low level, lower reagent costs, and significantly expand the detection capabilities of this biosensor. This article provides an overview of the current design and operation of the APDS. Certain sub-components of the ADPS are described in detail, including the aerosol collector, the automated sample preparation module that performs multiplexed immunoassays with confirmatory PCR, and the data monitoring and communications system. Data obtained from an APDS that operated continuously for 7 days in a major U.S. transportation hub is reported.
    Biosensors & Bioelectronics 05/2005; 20(10):1925-31. · 6.45 Impact Factor
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    ABSTRACT: This research analyzes the physical performance characteristics of the aerosol collectors of the autonomous pathogen detection system (APDS) that was recently developed by the Lawrence Livermore National Laboratory. The APDS is capable of continuous and fully autonomous monitoring for multiple airborne threat organisms and can be used as part of a monitoring network for urban areas and major public gatherings. The system has already been successfully tested with airborne Bacillus anthracis and Yersinia pestis biowarfare agents. The APDS aerosol collection system consists of a PM-style cap to remove large particles and a low-pressure drop virtual impactor preconcentrator positioned in front of a wetted-wall cyclone. The aerosol collectors operate at flow rates as high as 3750 l/min and collect airborne particles into 4 ml of liquid for subsequent detection. In our tests we determined the overall collection efficiency of the system by measuring the difference between inlet and outlet particle concentrations. The tests were performed with polydisperse oleic acid and monodisperse polystyrene latex (PSL) particles (0.6–3.1 µ m), and for three values of the major air flow rates in the virtual impactor (1760, 2530, and 3300 l/min), two values of the product, or cyclone, flow rates (375 and 450 l/min), and two different volumes of collection liquid (4 and 6 ml). We found that the cutoff size ( d 50 ) of the entire collection system varied from 1.5 to 2.0 µ m when collecting PSL particles, with 3.1 µ m PSL particles being collected with efficiency of approximately 85%. When collecting oleic acid particles the d 50 of the entire system varied from 1.1 to 1.6 µ m. The concentration rates of the aerosol collection system were found to increase with increasing overall collection flow rate and approached one million per minute at the highest tested flowrates. Such high concentrating rates and high air sample volumes make the APDS collection system highly suitable for detecting low concentrations of airborne pathogens.
    Aerosol Science and Technology 04/2005; 39(5):461-471. · 2.78 Impact Factor
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    ABSTRACT: The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii, and botulinum toxoid. By coupling highly selective antibody- and DNA-based assays, the probability of an APDS reporting a false positive is extremely low.
    Analytical Chemistry 02/2005; 77(1):284-9. · 5.82 Impact Factor
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    ABSTRACT: An automated sample preparation module, based upon sequential injection analysis (SIA), has been developed for use within an autonomous pathogen detection system. The SIA system interfaced aerosol sampling with multiplexed microsphere immunoassay-flow cytometric detection. Metering and sequestering of microspheres using SIA was found to be reproducible and reliable, over 24-h periods of autonomous operation. Four inbuilt immunoassay controls showed excellent immunoassay and system stability over five days of unattended continuous operation. Titration curves for two biological warfare agents, Bacillus anthracis and Yersinia pestis, obtained using the automated SIA procedure were shown to be similar to those generated using a manual microtiter plate procedure.
    Analytical Chemistry 08/2004; 76(13):3492-7. · 5.82 Impact Factor
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    ABSTRACT: The autonomous pathogen detection system (APDS) is an automated, podium-sized instrument that continuously monitors the air for biological threat agents (bacteria, viruses, and toxins). The system has been developed to warn of a biological attack in critical or high-traffic facilities and at special events. The APDS performs continuous aerosol collection, sample preparation, and detection using multiplexed immunoassay followed by confirmatory PCR using real-time TaqMan assays. We have integrated completely reusable flow-through devices that perform DNA extraction and PCR amplification. The fully integrated system was challenged with aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii and botulinum toxoid. By coupling highly selective antibody and DNA based assays, the probability of an APDS reporting a false positive is extremely low.
    Published in: Analytical Chemistry, n/a, n/a, October 12, 2004, In press. 05/2004;
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    ABSTRACT: Researchers at Lawrence Livermore National Laboratory are developing means to collect and identify fluid-based biological pathogens in the forms of proteins, viruses, and bacteria. To support detection instruments, we are developing a flexible fluidic sample preparation unit. The overall goal of this Microfluidic Module is to input a fluid sample, containing background particulates and potentially target compounds, and deliver a processed sample for detection. We are developing techniques for sample purification, mixing, and filtration that would be useful to many applications including immunologic and nucleic acid assays. Sample preparation functions are accomplished with acoustic radiation pressure, dielectrophoresis, and solid phase extraction. We are integrating these technologies into packaged systems with pumps and valves to control fluid flow and investigating small-scale detection methods
    Microtechnologies in Medicine & Biology 2nd Annual International IEEE-EMB Special Topic Conference on; 02/2002
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    ABSTRACT: The Autonomous Pathogen Detection System (APDS) 1 is a stand-alone pathogen detection system capable of rapid, continuous, low cost environmental monitoring of multiple airborne biological threat agents. Its basic design comprises aerosol sampling, in-line sample preparation, multiplex detection and identification immunoassays, and orthogonal, multiplexed PCR (nucleic acid) amplification and detection. Its primary application is to warn civilians and emergency preparedness personnel of a terrorist attack, the same system could also have a role in protecting military personnel from biological warfare attacks. APDS instruments can be used at high profile events such as the Olympics for short-term, intensive monitoring or more permanent installation in major public buildings or transportation nodes. All of these units can be networked to a single command center so that a small group of technical experts could maintain and respond to alarms at any of the sensors. The APDS has several key advantages over competing technologies: (1) the ability to measure up to 100 different agents and controls in a single sample, (2) the flexibility and ease with which new bead-based assays can be developed and integrated into the system, (3) the presence of an orthogonal, real-time detection module for highly sensitive and selective nucleic acid amplification and detection, (4) the ability to use the same basic system components for multiple deployment architectures, and (5) the relatively low cost per assay (<2 per 10-plex or2 per 10-plex or 0.20 per assay) and minimal consumables.
    01/1970: pages 67-75;
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    ABSTRACT: A dispensing system delivers a precise amount of fluid for biological or chemical processing and/or analysis. Dispensing means moves the fluid. The dispensing means is operated by a pneumatic force. Connection means delivers the fluid to the desired location. An actuator means provides the pneumatic force to the dispensing means. Valving means transmits the pneumatic force from the actuator means to the dispensing means.
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    ABSTRACT: An Autonomous Pathogen Detection System (APDS) unit is an automated, podium-sized system that monitors the air for all three biological threat agents (bacteria, viruses, and toxins). The system has been developed under the auspices of the U. S. Department of Energy and Department of Homeland Security by the University of California, Lawrence Livermore National Laboratory (LLNL) to protect people in critical or high-traffic facilities and at special events. The system performs continuous aerosol collection, sample preparation, and multiplexed biological tests using advanced immunoassays as the primary screen. Over ten agents are assayed at once, and results are reported hourly. R&D work this year focused on incorporating polymerase chain-reaction (PCR) techniques for detecting DNA as confirmation of immunoassay positives. The primary objective of the Dugway testing was to demonstrate the APDS with immunoassay identification and PCR confirmation of bacteria. A secondary objective was to demonstrate immunoassay identification of a protein toxoid (denatured toxin) aerosol release. A total of 12 agent trials were conducted over 14 days of testing, for a total of four work weeks at Dugway. Both testing objectives were achieved with multiple releases and clear identifications. The APDS was shown to be effective for identifying aerosolized Bacillus anthracis, Yersinia pestis, Bacillus globigii, and botulinum toxoid. The two areas for improvement were operational as opposed to hardware-related. The first was slowing the PCR thermal cycling to achieve stronger signals, which was demonstrated during the later phases of testing. The second area is to improve the parameters for autonomous PCR triggering; this is one of the focuses of the upcoming year's work.