CHAPTER 2: REVIEW OF ENDOSPORE DETECTION TECHNOLOGY
2.9 Spore detection instruments
Yung et al. have developed a fully automated anthrax smoke detector (ASD)93-95. The ASD is intended to serve as a cost-effective front-end monitor for anthrax surveillance systems. The principle of operation is based on measuring airborne endospore concentrations, where a sharp concentration increase signals an anthrax attack. The ASD features an air sampler, a thermal lysis unit, a syringe pump, a time-gated spectrometer, and endospore detection chemistry comprised of dipicolinic acid (DPA)-triggered terbium ion (Tb3+) luminescence. Anthrax attacks were simulated using aerosolized B.
atrophaeus spores in fumed silica, and corresponding Tb-DPA intensities were monitored as a function of time and correlated to the number of airborne endospores collected. A concentration dependence of 102-106 spores mg-1 of fumed silica yielded a dynamic range of 4 orders of magnitude and a limit of detection of 16 spores L-1 when 250 L of air were sampled. Simulated attacks were detected in less than 15 minutes. A comparison between ASD and PCR-based detectors performance has been summarized in Figure 2.2.
Eversole et al. have demonstrated a prototype single particle fluorescence analyzer (SPFA) to simultaneously monitor ambient concentrations of both biological and nonbiological aerosols96. The instrument pulls air through a nozzle at approx. 300 liters per minute and monitors particles between 1 and 10 µm in diameter in situ using laser- induced excitation and detection of two specific bands: UV (300–400 nm) and visible (400–600 nm). Discrimination between biological and nonbiological aerosols is effected
through comparison of these emission intensities to a calibrated reference. The latest outdoor field testing of the instrument resulted in a detection probability of 87% for the target aerosols ova albumin, MS-2 phage, Erwinia herbicola vegetative cells and B.
subtilis spores, with airborne concentrations of 5 particles per liter. Further, the absolute quantitative detection efficiencies for individual biological aerosols all averaged over 70%, with the measurement response time proportional to the particle measurement rate.
Agranovski et al. have devised an instrument for real-time continuous monitoring of bioaerosols called the Ultraviolet Aerodynamic Particle Size Spectrometer (UVAPS)97. The instrument is capable of providing time-of-flight, light scattering and fluorescence measurements for particles ranging from 0.5 to 15 µm in diameter. Aerosolized spores (Bacillus subtilis), vegetative cells (B. subtilis, Pseudomonas fluorescens) and non- bacterial elements (NaCl, latex, peptone water, nutrient agar) were all used to test the selectivity, sensitivity, efficiency and limit of detection of the instrument. Results indicated that the UVAPS was able to detect bacterial spores with limited capability, and only the B. subtilis vegetative cells produced a strong fluorescent signal, with a limit of detection of approximately 107 particles m-3. Further, strong false positives from nonbacterial elements were also observed. Particle counting efficiency depends on particle concentration, and has a “saturation” level of 106 particles m-3.
Another instrument designed and tested by McBride et al., called the fully autonomous pathogen detection system (APDS), is capable of continuous monitoring for bioaerosols98. The instrument is composed of an aerosol collector, a fluidics module for sample preparation and immunoassay detection using a flow cytometer. The immunoassay uses a sandwich format, where antigen-specific antibodies immobilized on
beads bind the analyte, which is then detected using secondary fluorescently labeled antibodies. This analysis requires 60 s to complete. The system was tested with release of B. anthracis and Yersinia pestis, both of which it was able to detect with no false positives. Plus, with a collection interval of 30 to 60 min and a capability of continuous unattended operation for 8 days, the system can be easily integrated into a central security network. The system will ultimately utilize an orthogonal detection approach including antibody-based (immunoassays) and nucleic acid-based (PCR) assays to reduce false positives.
An instrument developed by Cheng et al., is used to aerosolize bacterial spores to improve fluorescence detection of aerosols99. As opposed to using only single-excitation wavelength approaches, the authors utilize multiple wavelengths of excitation and fluorescence detection. The system, including an aerosol generator, chamber, aerosol monitoring instrumentation and laser-induced fluorescence detection system, was able to obtain fluorescence measurements on Escherichia coli, Staphylococcus aureus, Bacillus subtilis var. niger, and Bacillus thuringiensis, but was not able to distinguish between them.
Brosseau et al., utilize the autofluorescent chemicals naturally found in bacteria (spore and vegetative cells) and fungi, such as riboflavin, nicotinamide adenine dinucleotide phosphate (NADPH) and tryptophan, to detect biological aerosols100. Nebulized spores and bacterial cells of Bacillus subtilis subsp. niger, as well as various other bacteria and even fungal spores were evaluated using an Ultraviolet Aerodynamic Particle Sizer (UV-APS). It was found that fluorescence appears to be species-dependent, but identification of bioaerosols was made difficult due to high variability.
Scientists at Lawrence Livermore National Laboratory have also invented a stand- alone system for rapid, continuous monitoring of multiple airborne biological threat agents in the environment. This system, the autonomous pathogen detection system (APDS), acts as a biological “smoke alarm” and is targeted for domestic applications in which the public is at high risk of exposure to covert releases of bioagent (such as mass transit, office complexes, and convention centers), and as part of a monitoring network for urban areas and major gatherings.
The APDS is completely automated, offering aerosol sampling, in-line sample preparation fluidics, multiplex flow cytometer detection and identification assays, and orthogonal, flow-through PCR (nucleic acid) amplification and detection. For the flow- cytometry subsystem, small “capture” beads 5 µm in diameter are coated with antibodies specific to the target pathogens. The beads are color-coded according to which antibodies they hold. Once the pathogens attach to their respective antibodies, more antibodies (labeled with a fluorescent dye), are added to the mixture. A labeled antibody will stick to its respective pathogen, creating a sort of bead sandwich—antibody, pathogen, and labeled antibody. The beads flow one by one through a flow cytometer, which illuminates each bead in turn with a laser beam. Any bead with labeled antibodies will fluoresce. The system can then identify which agents are present, depending on the color of the capture bead.
Advantages include:(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) low false-positive and false-negative detection due to the presence of two orthogonal detection methods; (4) the ability to use
the same basic system components for multiple deployment architectures; (v) the relatively low cost per assay and minimal consumables.