John M. Dzenitis

Underwater application of quantitative PCR on an ocean mooring.

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.

Environmental Monitoring for Biological Threat Agents Using the Autonomous Pathogen Detection System with Multiplexed Polymerase Chain Reaction

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 instruments 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.

LAVA: An Open-Source Approach To Designing LAMP (Loop-Mediated Isothermal Amplification) DNA Signatures

BackgroundWe 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.ResultsLAVA 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.ConclusionsWe 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/.

Performance Characteristics of the Aerosol Collectors of the Autonomous Pathogen Detection System (APDS)

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 (d50 ) 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 d50 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.

Microfluidic tools for biological sample preparation

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.

An overview of target and diagnostic alignment at the National Ignition Facility

The National Ignition Facility (NIF) is a 192-beam high energy laser designed for Inertial Confinement Fusion (ICF), and High Energy Density (HED) and basic science experiments. In order to achieve ignition with an ICF target, the beam and target alignment must be performed within very tight specifications. At the same time, in order to be able to conduct the wide range of HED and basic science experiments, the facility must be able to meet the tight tolerances for both main and offset backlighter beams and targets. To diagnose the ignition event, many different target diagnostics are employed, including optical, x-ray, and nuclear diagnostics. These target diagnostics must also be positioned accurately and reliably within very tight specifications in order to ensure good data is acquired. In this paper, we describe the strategy for beam, target, and diagnostic alignment at NIF.

The 27.3 meter neutron time-of-flight system for the National Ignition Facility

One of the scientific goals of the National Ignition Facility (NIF) at Lawrence Livermore National Laboratory, Livermore CA, is to obtain thermonuclear ignition by compressing 2.2 mm diameter capsules filed with deuterium and tritium to densities approaching 1000 g/cm3 and temperatures in excess of 4 keV. Thefusion reaction d + t → n + a results in a 14.03 MeV neutron providing a source of diagnostic particles to characterize the implosion. The spectrum of neutrons emanating from the assembly may be used to infer the fusion yield, plasma ion temperature, and fuel areal density, all key diagnostic quantities of implosion quality. The neutron time-of-flight (nToF) system co-located along the Neutron Imaging System line-of-site, (NIToF), is a set of 4 scintillation detectors located approximately 27.3 m from the implosion source. Neutron spectral information is inferred using arrival time at the detector. The NIToF system is described below, including the hardware elements, calibration data, analysis methods, and an example of its basic performance characteristics.

On-chip laser-induced DNA dehybridization.

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 microarrays 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.

Apds, a Network-Ready, Broad Spectrum, Environmental Pathogen Detection System

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 (<

Performance characteristics of the neutron imaging diagnostic at NIF

2 per 10-plex or