Environmental Monitoring for Biological Threat Agents Using the

In September and October of 2001, letters sent through the U.S. mail system containing small amounts of Bacillus anthracis spores caused five deaths a...
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Environmental Monitoring for Biological Threat Agents Using the Autonomous Pathogen Detection System with Multiplexed Polymerase Chain Reaction John F. Regan, Anthony J. Makarewicz, Benjamin J. Hindson, Thomas R. Metz, Dora M. Gutierrez, Todd H. Corzett, Dean R. Hadley, Ryan C. Mahnke, Bruce D. Henderer, John W. Breneman IV, Todd H. Weisgraber, and John M. Dzenitis* Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94550 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 onetenth the time of manual filter collection and analysis. In September and October of 2001, letters sent through the U.S. mail system containing small amounts of Bacillus anthracis spores caused five deaths and several cases of severe inhalation anthrax.1 To deter and reduce the impact of a widespread biological agent release, the U.S. Department of Homeland Security (DHS) established the BioWatch program to perform environmental air monitoring in many of the nation’s urban centers for the presence of aerosolized pathogens.2 This nationwide surveillance system uses distributed aerosol collectors to capture airborne particles onto removable dry filters that are transported daily to Laboratory Response Network (LRN) laboratories for * To whom correspondence should be addressed. Phone: (925) 422-6695. Fax: (925) 422-4100. E-mail: [email protected]. (1) Read, T. D.; Salzberg, S. L.; Pop, M.; Shumway, M.; Umayam, L.; Jiang, L.; Holtzapple, E.; Busch, J. D.; Smith, K. L.; Schupp, J. M.; Solomon, D.; Keim, P.; Fraser, C. M. Science 2002, 296, 2028–2033. (2) Shea, D. A., Lister, S. A. The BioWatch Program: Detection of Bioterrorism, Congressional Research Service Report No. RL 32152, published 2003. Accessed online at http://www.fas.org/spg/crs/terror/RL32152.html on March 31, 2008.

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analysis. In an effort to improve the surveillance capabilities of the BioWatch program and to reduce the program’s operational costs, DHS continues to evaluate new technologies. The majority of biodetection technologies under development that may be considered for use in biothreat surveillance focus on either nucleic acid3-6 or protein detection.7,8 Some of these technologies have been integrated into field-deployable autonomous environmental surveillance instruments including the following: the Joint Biological Point Detection System,9 the Biohazard Detection System,10 the Single-Particle Aerosol Mass Spectrometry System,11,12 the BioFlash Biological Identifier, which incorporates technology developed at the MIT Lincoln Laboratory,13 and the Autonomous Pathogen Detection System (APDS).14 Such systems are designed to provide earlier detection than is possible with manual surveillance methodologies, which is crucial for mitigating consequences and saving lives. Furthermore, the automation of surveillance assays can reduce the operational costs per sample by more than one order of magnitude by removing the labor associated with sample transportation and manual analysis. (3) Monis, P. T.; Giglio, S. Infect. Genet. Evol. 2006, 6, 2–12. (4) Ivnitski, D.; O’Neil, D. J.; Gattuso, A.; Schlicht, R.; Calidonna, M.; Fisher, R. Biotechniques 2003, 35, 862–869. (5) Chan, E. Y. Mutat. Res. 2005, 573, 13–40. (6) Tost, J.; Gut, I. G. Clin. Biochem. 2005, 38, 335–350. (7) Fischer, N. O.; Tarasow, T. M.; Tok, J. B. Analyst 2007, 132, 187–191. (8) Ngundi, M. M.; Kulagina, N. V.; Anderson, G. P.; Taitt, C. R. Expert Rev. Proteomics 2006, 3, 511–524. (9) Christie, T. P., Director, Operational Test & Evaluation, Annual Report, FY 2003. Accessed online at http://www.globalsecurity.org/military/ library/budget/fy2003/fy03_DOTE_Annual_Report.pdf on April 12, 2008. (10) Meehan, P. J.; Rosenstein, N. E.; Gillen, M.; Meyer, R. F.; Kiefer, M. J.; Deitchman, S.; Besser, R. E.; Ehrenberg, R. L.; Edwards, K. M.; Martinez, K. F. MMWR Recommendations Rep. 2004, 53, 1–12. (11) Fergenson, D. P.; Pitesky, M. E.; Tobias, H. J.; Steele, P. T.; Czerwieniec, G. A.; Russell, S. C.; Lebrilla, C. B.; Horn, J. M.; Coffee, K. R.; Srivastava, A.; Pillai, S. P.; Shih, M. T.; Hall, H. L.; Ramponi, A. J.; Chang, J. T.; Langlois, R. G.; Estacio, P. L.; Hadley, R. T.; Frank, M.; Gard, E. E. Anal. Chem. 2004, 76, 373–378. (12) Steele, P. T.; Farquar, G. R.; Martin, A. N.; Coffee, K. R.; Riot, V. J.; Martin, S. I.; Fergenson, D. P.; Gard, E. E.; Frank, M. Anal. Chem. 2008, 80, 4583– 4589. (13) Rider, T. H.; Petrovick, M. S.; Nargi, F. E.; Harper, J. D.; Schwoebel, E. D.; Mathews, R. H.; Blanchard, D. J.; Bortolin, L. T.; Young, A. M.; Chen, J.; Hollis, M. A. Science 2003, 301, 213–215. (14) Hindson, B. J.; Makarewicz, A. J.; Setlur, U. S.; Henderer, B. D.; McBride, M. T.; Dzenitis, J. M. Biosens. Bioelectron. 2005, 20, 1925–1931. 10.1021/ac801125x CCC: $40.75  2008 American Chemical Society Published on Web 09/03/2008

The APDS is a fully automated instrument that captures aerosolized particles into liquid and performs biothreat surveillance assays at programmed intervals during 7 days of continuous unattended operation. Earlier-generation APDS instruments used sequential injection analysis15 and zone fluidics16 to perform multiplexed immunoassays17,18 using Luminex microsphere array technology in a flow-through system; the next-generation instrument added the capability of confirming immunoassay results with real-time polymerase chain reaction (PCR) analysis.19 The realtime PCR-capable model utilized a flow-through thermal cycler20 and performed nucleic acid extraction using a microfabricated silica chip.21 Early- and late-generation instruments were challenged in Biological Safety Level (BSL) 3 and 2 chambers at the Dugway Proving Ground with live and inactivated aerosolized agents including B. anthracis and Yersinia pestis. The newest generation APDS instrument presented in this report performs a broad-spectrum, multiplexed PCR assay for the detection of several biothreat agents. The advantages of incorporating this assay include multiloci detection to reduce the possibility of erroneous results and the consolidation of several assays into one multiplexed assay, which reduces the operational complexity and cost of performing biothreat surveillance. We demonstrate the ability of this new multiplexed PCR module of the APDS instrument to detect B. anthracis and Y. pestis samples and compare automated APDS generated data to manual laboratory generated results using real-time PCR assays. We also demonstrate the system’s specificity and reliability during 74 days of field operation in a mass-transit subway station. MATERIALS AND METHODS The APDS shown in Figure 1A is a stand-alone device that collects particles from the air to provide sample, performs and analyzes multiplexed PCR assays, and maintains communications with a central database. Detailed descriptions of the system’s individual components, the multiplexed PCR assay, and experimental procedures and are provided below. Aerosol Collector. The aerosol collector of the multiplexed PCR APDS unit is an upgraded version of the previously described collector.17 Air is drawn into the system through a stack on the roof of the APDS enclosure at a rate of 1700 L of air/min (Figure 1A). The air passes through a series of filter screens, magnetic traps, and momentum-based particle separators that selectively remove unwanted particles (e.g., ferromagnetic steel dust present (15) Ruzicka, J. a.; M, G. D. Anal. Chim. Acta 1990, 237, 329–343. (16) Ahlfors, C. E.; Marshall, G. D.; Wolcott, D. K.; Olson, D. C.; Van Overmeire, B. Clin. Chim. Acta 2006, 365, 78–85. (17) McBride, M. T.; Masquelier, D.; Hindson, B. J.; Makarewicz, A. J.; Brown, S.; Burris, K.; Metz, T.; Langlois, R. G.; Tsang, K. W.; Bryan, R.; Anderson, D. A.; Venkateswaran, K. S.; Milanovich, F. P.; Colston, B. W., Jr Anal. Chem. 2003, 75, 5293–5299. (18) Hindson, B. J.; Brown, S. B.; Marshall, G. D.; McBride, M. T.; Makarewicz, A. J.; Gutierrez, D. M.; Wolcott, D. K.; Metz, T. R.; Madabhushi, R. S.; Dzenitis, J. M.; Colston, B. W., Jr Anal. Chem. 2004, 76, 3492–3497. (19) Hindson, B. J.; McBride, M. T.; Makarewicz, A. J.; Henderer, B. D.; Setlur, U. S.; Smith, S. M.; Gutierrez, D. M.; Metz, T. R.; Nasarabadi, S. L.; Venkateswaran, K. S.; Farrow, S. W.; Colston, B. W., Jr.; Dzenitis, J. M. Anal. Chem. 2005, 77, 284–289. (20) Belgrader, P.; Elkin, C. J.; Brown, S. B.; Nasarabadi, S. N.; Langlois, R. G.; Milanovich, F. P.; Colston, B. W., Jr.; Marshall, G. D. Anal. Chem. 2003, 75, 3446–3450. (21) Hindson, B. J.; Gutierrez, D. M.; Ness, K. D.; Makarewicz, A. J.; Metz, T. R.; Setlur, U. S.; Benett, W. B.; Loge, J. M.; Colston, B. W., Jr.; Francis, P. S.; Barnett, N. W.; Dzenitis, J. M. Analyst 2008, 133, 248–255.

in the air of subway stations) prior to entering a wetted-wall cyclone collector (Research International SASS 2000), which selectively captures particles in the 1-10-µm size range. The volume of water in the cyclone is maintained at 4 mL using a liquid inventory sensor and onboard water to make up for evaporation. At the end of the collection period, the cyclone is turned off to allow a syringe pump to pull the particulate-filled fluid into the APDS fluidics module to serve as a sample for testing and archiving. After the cyclone is drained, it is washed, rinsed, and refilled with fresh deionized water and then turned back on to resume particulate collection. Fluidics Module. The custom APDS fluidics module (Figure 1B) responsible for performing multiplexed PCR assays is based on the FloPro 4P by Global FIA (Fox Island, WA) and is composed of three multiposition valves, each with 14 ports (VICI, Houston, TX), and a XP3000 syringe pump (Cavro, Inc., Sunnyvale, CA) equipped with a 1-mL barrel syringe. Perfluoroalkoxy (PFA) Teflon tubing (Cole-Parmer, Vernon Hills, IL, Upchurch Scientific, Oak Harbor, WA, and Medical Extrusion Technologies Inc., Murrieta, CA) is used to connect the valves and syringe pumps to the other components of the system. All tubing associated with performing the multiplexed assays is 1/16-in. o.d. with i.d.s of 0.01, 0.02, 0.03, and 0.045 in. The fluidics module is used to perform sequential injection analysis,15 which is the coordinated movement of fluid through tubing using a syringe pump and multiposition valves. More specifically, the system performs zone fluidics,16 which is the sequential aspiration of test reactants and air into tubing to form a reactant bolus or “zone” surrounded by air. The reactant liquid may be combined with other reagents and moved to different locations within the fluidic manifold to complete each step of a desired assay. Flow-Through Thermal Cycler. The APDS platform includes a flow-through thermal cycler (Figure 1B), which is used for PCR amplification and the later hybridization of the amplified PCR products to pathogen-specific microspheres. The heater is an improved version of the previously described silicon-etched chamber20 and is made of a 48-mm copper sleeve braced against a series of resistors to provide heating. The copper sleeve is longer than the previous silicon-etched chamber, enabling the flowthrough tube to accommodate a larger volume (48 uL) of fluid and heat the fluid more uniformly and reliably. Temperature control is achieved by monitoring a resistance temperature detector feedback loop and accordingly altering the voltage across the resistors to reach the desired temperature. A fan speeds the cooling of the copper sleeve during temperature ramp-down. The system’s computer communicates with and controls the flowthrough thermal cycler via a data acquisition card (National Instruments, Austin, TX). Microsphere Reservoir. Microspheres for the multiplexed assay are held in a custom-made, light-protected, 4-mL poly(methyl methacrylate) reservoir. Microspheres are kept suspended by a stainless steel impeller driven at 200 rpm by a Minimotor (Faulhaber, Switzerland). Microsphere Sequestering Cell. The microsphere sequestering cell (Global FIA, Inc.)18 is used to retain microspheres during reporter incubation and washes. It is connected to multiposition valves 1 and 3 (Figure 1B) by two terminal lines and two peripheral lines. The terminal lines are associated with the lumen Analytical Chemistry, Vol. 80, No. 19, October 1, 2008

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Figure 1. Autonomous pathogen detection system (APDS) encased in a tamper-resistant, environmentally controlled enclosure (A). The aerosol collector, uninterruptible power supply, fluidic manifold, and the Luminex LX100 analyzer are visible from top to bottom, respectively. Air is drawn into the circular duct at the top of the instrument for multiplexed analysis. Internet connectivity and power are supplied to the back of the instrument. A layout of the fluidic manifold is provided in a schematic diagram (B), illustrating the connections between the aerosol collector, syringe pump, valves, flow-through thermal cycler, sequestering cell, Luminex analyzer, reagents, and waste receptacle.

of a microporous polypropylene tube within the sequestering cell. The peripheral lines are associated with a shell surrounding the porous tube. The terminal and peripheral sides are completely separate, communicating only through the porous wall of the tube. The tube has a mean pore size of 0.2 µm and has been treated with fluorosurfactant Zonyl FSN (0.05% v/v, Dupont) to improve wetting. Fluid flow through the sequestering cell can occur through three paths, vertically through the lumen of the tube, vertically between the porous tube and the inner side of the outer shell, and diagonally through the porous tube membrane in either direction. Microspheres (5.6 µm in diameter) can be retained within the sequestering cell by diagonally pumping fluid across the porous membrane. Retained microspheres can be incubated with reporter molecules and washed prior to liberating the microspheres, which occurs by pumping fluid through the membrane in the opposite direction. Luminex Analyzer. The fluidics module is connected to a Luminex LX100 analyzer (Luminex Corp., Austin, TX), equipped with 532- and 635-nm lasers used in detecting reporter molecules (e.g., phycoerythrin) and classifying microspheres, respectively. The Luminex analyzer is able to accurately distinguish 100 different microsphere classes, at rates exceeding 500 microspheres/s. The analyzer interrogates 80 µL of microspheres that 7424

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have been liberated from the sequestering cell and calculates the median fluorescence intensity (MFI) of each microsphere class. Thresholds for each microsphere class were empirically determined using plain water samples and water samples spiked with biothreat agents, in the presence and absence of confounding material. Reagents. Streptavidin-phycoerythrin (SAPE) (Caltag Laboratories, Burlingame, CA, or Prozyme, San Leandro, CA), a fluorescent reporter used to determine the presence of amplified product, is held in a 15-mL polypropylene tube. Reagents used to decontaminate and condition the instrument’s tubing and components include the following: 100 mM Tris pH 8.0, 200 mM NaCl, 0.05% Triton-X 100 buffer (Teknova, Inc., Hollister, CA); 0.6 M sodium citrate, 0.1 M MOPS, pH 7.5 (Pierce Biotechnology, Inc., Rockford, IL); Tris-EDTA pH 8.0 (Teknova, Inc., Hollister, CA); and 1.3% sodium hypochlorite. These reagents were found to be sufficiently stable at 23 °C for one week of operation without a significant loss in instrument sensitivity or specificity (data not shown). System Control. A Dell Latitude D600 laptop containing LLNL’s in-house Instrument Scripting and Data Acquisition Terminal (ISDAT) software (Version 2.1.8), created using LabVIEW Version 7.1 (National Instruments), controls the system’s

components through routines composed of configurable commands. These routines orchestrate the cycle of analysis through aerosol sample collection, sample transfer, multiplexed PCR assay execution, flow analyzer analysis, and system cleanup. The ISDAT software is also used to configure assay and instrument parameters, including: thermal cycler heating profiles, microsphere MFI thresholds, the selection of screened biothreat agents and the associated microsphere classes, and nominal operating parameters. We also developed a communications and control system that enables remote viewing of instrument operations via a secure Internet connection. Instruments upload data into a central database that may be remotely viewed to assess assay results and the overall system operation. The communications software may also be used to perform remote operation of field-deployed instruments for troubleshooting purposes. Enclosure. All of the APDS material is encased in a custom, tamper-resistant, outdoor enclosure (DDB Unlimited, Inc., Wynnewood, OK). The internal air temperature is maintained at 23 °C in hot and cold external environments with an Advantage Series RP17 air conditioner (Kooltronic, Inc., Pennington, NJ) and a DAH8001B electric heater (Hoffman Enclosures, Inc., Anoka MN). Multiplexed Assay. The primers and probes that make up the biothreat signatures of the multiplexed assay were designed by the Pathogen Informatics Group at Lawrence Livermore National Laboratory (LLNL, Livermore, CA), the Centers for Disease Control and Prevention (CDC, Atlanta, GA), and the Naval Medical Research Center (NMRC, Bethesda, MD). The multiplexed panel exceeds a 20-plex assay and screens for multiple biothreat agents including, but not limited to, B. anthracis and Y. pestis. The instrument performs each of the steps involved in a multiplexed PCR assay (Figure 2). First, a sample (6 µL), either acquired from the aerosol collector or directly introduced onto the fluidic manifold, is mixed with primers (8 µL) and enzyme/ buffer mix (16 µL) and delivered to the thermal cycler for amplification. The multiplexed primer mix includes each primer at 1.5 µM (final concentration of 0.4 µM) and 5 pg of positive control DNA (Erwinia herbicola template) per 8-µL volume. The enzyme/buffer mix is composed of 3 µL of 10 × LightCycler FastStart DNA Master HybProbe Mix (Roche Applied Science), 4.8 µL of 25 mM MgCl2, and 8.2 µL of PCR-grade water. The reaction mixture is transported to the flow-through thermal cycler where it is heated to 95 °C for 2 min, followed by 35 cycles of 94 °C for 15 s, 60 °C for 30 s, and 72 °C for 15 s. Amplified PCR product (5 µL) is then mixed with the microspheres (25 µL) and returned to the thermal cycler for hybridization, which involves denaturing the PCR product at 94 °C for 2 min, followed by annealing of the PCR product to probe-coupled microspheres at 55 °C for 5 min. In the presence of target nucleic acid, specific biotinylated forward primers are extended during PCR amplification, providing the necessary sequence to facilitate hybridization to the probe-coupled microspheres. The hybridized microspheres are transported to the sequestering cell to be incubated with SAPE, which binds to the biotin molecule(s) on the forward primers.

Figure 2. Flow diagram of the APDS multiplexed PCR assay process. A liquid sample is retrieved from the wetted-wall cyclone and combined with enzyme and primers to complete the multiplexed PCR reaction mixture, which is delivered to the thermal cycler for amplification. Microspheres coupled to pathogen-specific nucleic acid sequences are combined with the amplified products and returned to the thermal cycler where hybridization of the PCR products to the microspheres occurs. Hybridized microspheres are labeled with SAPE in the sequestering cell and washed before being delivered to the flow analyzer to determine the MFI associated with each microsphere class. The system then cleans itself and immediately repeats the process.

a. xMAP Technology. The presence or absence of amplified material in the multiplexed PCR reactions is determined using xMAP microsphere array technology (Luminex Corp.), which has been previously described.22-24 Briefly, xMAP liquid arrays include a choice of 100 different microsphere classes, determined by the ratio of red and near-infrared dyes impregnated into the polystyrene microspheres. The different ratios impart unique spectral addresses that can be discriminated by the analyzer’s optical detection unit associated with the 635-nm laser. Each microsphere can be functionalized by coupling a probe to the microsphere’s surface as previously described. Microspheres of a particular class are incubated with 0.05 mg of 1-ethyl-3-(3dimethylaminopropyl) carbodiimide hydrochloride (EDC, Pierce Biotechnology, Rockford, IL) in the presence of a specific probe. The EDC catalyzes the covalent linkage between an ester molecule found on the microsphere’s surface and a phosphoramidite group found at the 5′ end of each probe. The phosphoramidite group is separated from the probe sequence by an 18-atom hexaethylene glycol spacer. b. Assay Controls. The performance of the instrument and assay is monitored through four different controls. Negative control (NC) microspheres are coupled to a unique sequence not found in the terrestrial environment. NC serves as a control for nonspecific DNA amplification, since this sequence should not be present in aerosolized samples. The fluorescence control (FC) and instrument (22) Fulton, R. J.; McDade, R. L.; Smith, P. L.; Kienker, L. J.; Kettman, J. R., Jr Clin. Chem. 1997, 43, 1749–1756. (23) Wilson, W. J.; Erler, A. M.; Nasarabadi, S. L.; Skowronski, E. W.; Imbro, P. M. Mol. Cell. Probes 2005, 19, 137–144. (24) McBride, M. T.; Gammon, S.; Pitesky, M.; O’Brien, T. W.; Smith, T.; Aldrich, J.; Langlois, R. G.; Colston, B.; Venkateswaran, K. S. Anal. Chem. 2003, 75, 1924–1930.

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control (IC) are modifications of NC, where either select thymine residues are biotinylated or the 3′ end of the unique sequence is coupled to a synthetic fluorophore, respectively. The biotin molecules incorporated into the unique sequence of FC provide sites for SAPE binding, allowing this coupled microsphere class to serve as a control for the addition of reporter molecules, whereas the coupled synthetic fluorophore of IC enables this coupled microsphere class to serve as a control for the integrity of the analyzer’s reporter optics. In addition to these controls, the positive PCR control (PC) is a microsphere class to which a sequence segment from the genome of E. herbicola is coupled. The multiplexed primer mix includes both primers and 5 pg of the control genomic E. herbicola DNA (per reaction), providing the necessary components for successful PCR amplification. The amount of control genomic DNA included in the assay was minimized to prevent excessive reagent competition with other pathogen-specific primer pairs, while also allowing for reliable amplification that is sensitive to the presence of PCR inhibitors. c. Assay Analysis. Signatures that return MFI values exceeding predetermined thresholds are called signature reactive events, on the condition that the four associated controls are within their predetermined ranges. The presence of a single signature reactive event does not indicate the sample is positive for the targeted organism, as it is possible for nonspecific amplification or binding of biotinylated products to the microspheres to occur. To protect against false alarms, each biothreat agent is represented by at least three different signatures (i.e., multiloci detection). The presence of multiple signatures allows for in-assay confirmation of results. At least two or more signatures targeting the same pathogen must exceed their thresholds to cause the system to issue a silent alert that requires manual review. An individual with detailed knowledge of the instrument and assay reviews data generated from alerts using a portable remote console and determines whether sufficient evidence exists to issue a response. Sample Preparation. Initial stocks of B. anthracis and Y. pestis were kindly provided by the CDC and cultured at LLNL. Vegetative B. anthracis cells were induced to sporulate and washed with cold water, whereas live Y. pestis cells were washed in normal saline. The concentration and viability of stocks were characterized using a Petroff-Hausser counting chamber and light microscopy to determine the counts per milliliter and by streaking aliquots onto agar Petri dishes to measure the colony-forming units per milliliter. To minimize changes in the integrity of genomic DNA and viability that can occur during storage of Y. pestis cells, fresh cultures were grown and counted by light microscopy for each day of testing. Stocks were diluted in water to the appropriate sample concentration prior to being directly entered into the manual laboratory or APDS benchtop processes described below. This version of APDS takes the particulate-filled fluid from the aerosol collector and introduces this material directly into a multiplexed PCR reaction, without prior nucleic acid extraction and purification. Manual Process. The manual laboratory process involved adding 500 µL of characterized aqueous agent samples to capped tubes containing 50 mg of a 106-600-µm glass bead mixture. Tubes were placed on a “bead-beater” instrument for 5 min of shaking and then placed on ice for 2 min. Sample tubes were centrifuged to separate the supernatant from the beads and particulate debris. Supernatants were collected and passed through 7426

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0.22-µm Ultrafree-MC centrifugal filter units (Millipore Corp., Bedford, MA) to remove remaining insoluble material. Filtrates were transferred to Micron YM-100, 100-kDa NMWL filter units (Millipore Corp.) and centrifuged. Filters were placed in new collection tubes and 400 µL of TE buffer was added. The filters were partially centrifuged and 100 µL of retentate was transferred to fresh Micron YM-100, 100-kDa NMWL filter units. Two additional 400-µL TE washes were performed, with changes in collection tubes, before filters were placed in fresh collection tubes and 400 µL of water was added to each filter top. Filters were partially centrifuged until 100 µL remained. Retentates were reclaimed from the filter tops using pipets and analyzed (5 µL) by real-time TaqMan PCR. APDS Process. Samples were introduced into an APDS benchtop system from one of two standard, screw-type vials mounted to the fluidics module via the same ports and tubing that would normally be used to introduce either an aerosol collector sample or a control sample. The benchtop system would then perform the standard multiplexed PCR procedure including the LX100 analysis and data transfer. APDS Field Operations. In the field, integrated APDS units perform seven environmental sample assays per day and are serviced once per week. The service includes: cleaning the aerosol collector; cleaning and calibrating the Luminex analyzer; reconditioning reagent pickup lines; replenishing molecular reagents; filling bulk fluid reservoirs; and inspecting and restarting the system. The system then operates in a continuous cycle without manual intervention. RESULTS AND DISCUSSION Demonstration of Detection Capabilities. To demonstrate the analytical performance of the APDS multiplexed PCR module, we first tested the liquid sample stream containing deionized water that would normally come from the aerosol collector to determine the background fluorescence level generated from each pathogenspecific microsphere in the absence of target (Figure 3, Exp. #1). As expected, the fluorescence levels of all the pathogen microsphere classes were low with MFI values below 100, while PC MFI values were consistently above 1000, indicating PCR amplification and detection occurred in these reactions. We then spiked the liquid sample stream with purified nucleic acids from B. anthracis and Y. pestis samples that were placed on the instrument in alternating order to verify the instrument’s ability to successfully detect these agents without significant cross-reactivity (Figure 3, Exp. #2). Both B. anthracis and Y. pestis samples were detected by each of the three BA and four YP signatures, respectively, demonstrating multiloci detection that would result in highconfidence data. The BA and YP signatures were only elevated in the presence of the respective nucleic acid markers from the respective organisms, with no significant increase observed in the MFI values from the other biothreat agents represented in the multiplexed panel. These data not only demonstrate repeatable detection of two different agents but also illustrate the successfulness of the clean-in-place procedure performed by the instrument at the conclusion of each assay, since no significant carryover contamination between samples was observed. After establishing the instrument’s ability to successfully perform multiloci detection of nucleic acid markers of targeted pathogens when provided purified genetic material, the instrument

Figure 3. Analytical demonstration of APDS detection capabilities. Three separate experiments are compiled including: Exp. #1, processing four consecutive water samples; Exp. #2, processing extracted and purified (Pur) B. anthracis (BA) and Y. pestis (YP) samples in alternating fashion; and Exp. #3, processing viable B. anthracis and Y. pestis samples followed by the same samples that were mechanically lysed by bead-beating. Panels A-D show MFI values for groups of signatures: panel A for B. anthracis signatures; panel B for Y. pestis signatures; panel C for selected additional biothreat signatures; and panel D for assay controls.

was then challenged with detecting aliquots of either B. anthracis spores or Y. pestis vegetative cells that were either viable or mechanically lysed (Figure 3, Exp. #3). Although many different combinations and concentrations of agents were tested, data from just one experiment that focuses on B. anthracis and Y. pestis samples of dilute concentration are shown to illustrate the effect that mechanical lysis has on the detection of minute quantities of these pathogens. For B. anthracis, the aliquot of viable spores provided sufficient levels of nucleic acid specific to the pathogen to be detected by the most sensitive BA signature (BA-1) without sample preparation, whereas the lysed sample was detected by all three BA signatures. Similarly, the aliquot of viable Y. pestis cells was detected by the two most sensitive YP signatures (YP-1 and YP-4), whereas the lysed sample was detected by all four YP signatures. These data suggest that lysis may improve the sensitivity of the automated multiplexed PCR process. Demonstration of Performance. In order for APDS instruments to be utilized as autonomous biothreat surveillance tools, it was necessary to characterize the sensitivity and specificity

Figure 4. Comparison of the performance of APDS multiplexed analysis to a manual laboratory analysis for the detection of different concentrations of B. anthracis (panel A) and Y. pestis (panel B) samples. The APDS process introduced unlysed liquid sample directly into a multiplexed PCR assay, whereas the manual laboratory process introduced extracted and purified nucleic acids into single-plex TaqMan assays. The MFI values for each of the B. anthracis and Y. pestis signatures are shown for each sample concentration tested, represented as Ct values from real-time PCR analyses.

relative to currently used manual surveillance methodologies. Currently, citywide surveillance for aerosolized biothreat agents is achieved by retrieving filters from aerosol collectors on a daily basis and processing them for biothreat agents. The collected filters are processed in laboratories, where they undergo a beadbeating protocol to lyse any captured pathogens and the extracted nucleic acid is purified before it is analyzed using single-plex TaqMan assays. To evaluate the ability of the APDS instrument to detect concentrations of viable agents near the detection limit for the manual laboratory process, agent samples were split and processed separately according to their respective methodologies; the APDS instrument enters unprocessed samples directly into multiplexed analysis, whereas the manual laboratory process first extracts and purifies the genetic material prior to TaqMan analysis. Fourteen viable B. anthracis spores samples were split and processed accordingly. The multiplexed signal (MFI values) of the three B. anthracis signatures (BA-1, BA-2, BA-3) were plotted using a real-time PCR cycle threshold (Ct) to characterize each of the 14 samples tested (Figure 4A). The automated multiplexed PCR process shows a semiquantitative response to different concentrations of B. anthracis as determined by the Ct values generated from the manual TaqMan analyses. Generally speaking, B. anthracis samples with Ct values less than 32 were fully Analytical Chemistry, Vol. 80, No. 19, October 1, 2008

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detected by multiplexed analysis, whereas samples with Ct values between 32 and 34 were fairly reliably detected by multireactive signatures, but not always. In contrast, samples with Ct values above 34 were not completely detected, although single reactive signatures were present in all but the least concentrated sample. A similar experiment was performed with viable Y. pestis cells (Figure 4B). Twelve fresh Y. pestis samples of different concentrations were analyzed by both described methodologies. Again, the automated multiplexed PCR process shows a semiquantitative response to detecting concentrations of viable Y. pestis cells as determined by the manual real-time PCR process. The YP-1 and YP-4 signatures were highly reactive, even with the more dilute samples, whereas the YP-2 and YP-3 signatures were less responsive to samples with Ct values higher than 32. Overall, unlysed Y. pestis concentrations that gave real-time Ct values less than 34 were detected by two or more signatures, whereas those samples with Ct values greater than 34 were unreliably detected due to the lack of additional reactive signatures to support the highly reactive YP-4 signature. In the event of a consequential bioterrorist attack, the spores or cells of the released biothreat agent would be concentrated by the aerosol collector of an appropriately positioned APDS. The analyzed sample would likely be significantly more concentrated than the samples shown in Figure 4 that yielded Ct values above 32, and in which case, these more concentrated samples generally yield multiloci actionable detection events that are independent of sample lysis or purification. Demonstration of Real-World Operation. To evaluate the specificity and performance of APDS with the incorporated aerosol collector in its intended operational environment, we installed an APDS unit on the platform of a high-traffic, mass-transit subway station. The system remained in continuous operation, sampling 1700 L of air/min for over 74 days, during which time, 493 aerosol samples were analyzed by the automated multiplexed PCR assay (Figure 5). Each of the 493 assays yielded control MFI values within the specified ranges, with the exception of sample 91 whose PC signal dropped to 135, which is below the acceptable lower limit of 200 for this control (Figure 5, panel D). The assay on the next sample returned an acceptable PC MFI value without intervention. The biothreat agent signatures shown in the other panels (A-C) have low baseline noise with the primary exceptions being BA-1 and YP-4 signatures, which are two of the more sensitive signatures in the multiplexed panel. The BA-1 signature records baseline values that are consistently higher than the other signatures, but the values are below the response of that signature to real agent shown in Figure 4, with the exception of the most dilute sample tested. The YP-4 signature is slightly less responsive than BA-1, but does include a single sample (227) with an MFI of 322, which exceeds the predetermined threshold for the signature. The requirement for multiple reactive signatures to deem a sample positive classifies this single reactive event as a negative sample, illustrating the importance of having multiple signatures per agent to prevent false positives. CONCLUSION We have developed an APDS instrument capable of performing automated multiplexed PCR assays for multiple biothreat agents. The instrument uses an extremely specific multiplexed assay that includes multiple signatures per agent to increase the certainty 7428

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Figure 5. Performance of APDS in a high-traffic subway station. The APDS unit performed 493 multiplex assays over a 74-day period. The MFI values for B. anthracis, Y. pestis, five selected additional biothreat agent signatures, and the control signatures are shown in panels A-D, respectively. The MFI values of the responsive BA-1 signature are below threshold for all samples tested, as are the other BA signatures. A single reactive event above threshold was observed for YP-4, but the lack of corroborating MFI signals from the three other YP signatures deemed this anomalous point a negative sample.

in a positive result and to maximize the specificity of the system. To cover the numerous possible threats, multiple biological agents are surveyed at once, causing the number of signatures tested per sample to be quite large. Multiplexing the PCR assay for biothreat surveillance offers several advantages over conventional real-time PCR assays, including: (1) reducing the operational complexity in terms of number of reactions and reagents, (2) reducing the cost due to less polymerase enzyme used, (3) decreasing the potential for reaction failure, and (4) increasing the confidence in generated results due to built-in assay controls. This APDS instrument is the result of years of federally funded research, development, testing, and evaluation. The integrated platform has been upgraded over time to perform single-plex immunoassays, multiplexed immunoassays, real-time PCR assays,

and now multiplexed PCR assays. Recently, the APDS instrument became the first actionable autonomous detector component of the U.S. Department of Homeland Security’s BioWatch program. LLNL is now working with an industrial partner on commercialization and expansion of the technology. Future work will focus on improving the instrument’s sensitivity by incorporating a sample extraction and purification module that is similar to the pillar chip technology incorporated in previously published versions of APDS,19,21 as well as extending the instrument’s detection capabilities by adding additional signatures to the multiplexed assay, and reducing the cost to purchase and operate the system. ACKNOWLEDGMENT We thank Staci Kane, Julie Perkins, Corey Chinn, and Jason Olivas for reagent preparation and testing in the laboratory; Candice Cook for assisting in maintaining laboratory and field-deployed units; Bill Benett, Chris Spadaccini, and Dean Urone for mechanical design work; Kris Montgomery and Julie Avila for testing with viable

biological threat agents; and Richard Meyer of the CDC Bioterrorism Rapid Response and Advanced Technology Laboratory for providing initial pathogen stocks. We thank the public health, facility, and law enforcement representatives for evaluating the APDS and providing helpful feedback before incorporating the improved system into their antiterrorism operations. The U.S. Department of Homeland Security is acknowledged as the sponsor of this work, under a “work for others” arrangement, issued under the prime contract for research, development, test, and evaluation services between the U.S. Department of Energy and LLNL. This work was performed under the auspices of the U.S. Department of Energy by Lawrence Livermore National Laboratory in part under Contract W-7405-Eng-48 and in part under Contract DE-AC52-07NA27344. J.F.R. and A.J.M. contributed equally to this work.

Received for review June 3, 2008. Accepted July 19, 2008. AC801125X

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