Technologies to Enable Autonomous Detection for BioWatch Ensuring Timely and Accurate Information for Public Health Officials: Workshop Summary (2014) / Chapter Skim
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Appendix G: White Paper 2: Nucleic-Acid Signatures at Three Levels of Readiness for BioWatch
Appendix G: White Paper 2: Nucleic-Acid Signatures at Three Levels of Readiness for BioWatch
Pages 155-172
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From page 155... ...
A white paper prepared for the June 25–26, 2013, workshop on Strategies for Cost-Effective and Flexible Biodetection Systems That Ensure Timely and Accurate Information for Public Health Officials, hosted by the Institute of Medicine's Board on Health Sciences Policy and the National Research Council's Board on Life Sciences. The author is responsible for the content of this article, which does not necessarily represent the views of the Institute of Medicine or the National Research Council.
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From page 156... ...
Numerous high-quality commercial off-the-shelf instruments exist that perform the functions of the individual steps, yet field-tested and proven integrated systems are few. It is a particular challenge in any automated fluidic system to make 100 percent reliable fluidic interconnections along with the necessary methods to transport, meter, and process samples and reagents so that the laboratoryproven assays that normally are carried out by trained personnel with pipettes, filters, and centrifuges perform just as well in the automated system.
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From page 157... ...
. Item 8 is also somewhat of a catchall, covering simple chemistries, such as the Boom capture/release of nucleic acids, but may also include emerging techniques, including even capture, isolation, and release of nucleic-acid regions of interest on either fixed or liquid microarrays (DuBose et al., 2013)
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From page 158... ...
Depending upon the details of the aerosol, it could happen that effectively every viable bacterium breathed in could be retained within the lungs. The infectious dose of some bacteria and viruses has been measured to be essentially a single viable bacterium or particle with infectious virions (Jones et al., 2005)
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FIGURE G-1 Block diagram of components of a notional next-generation system, including compo 159 nents and functions that may be incorporated in a staged manner.
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All of this is to say that it is not hard to envision a situation in which a potentially pathogenic bacteria or virus could be present at physiologically relevant concentrations, while remaining well below the background concentration of physically similar, nonpathogenic aerosolized bacteria and viruses. NUCLEIC-ACID SIGNATURES What comprises nucleic-acid signatures?
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From page 161... ...
PHAAs are developed and utilized to support public health actions involving the potential exposure of an individual or, more commonly, groups of individuals to biothreat materials such as Ba spores. PHAAs have high specificity, high sensitivity, and are highly robust to provide critical information on agent-specific confirmation and further characterization to support public health decisions such as initiating a national or local health alert warning, initiating a public health investigation, conducting risk assessments to support postexposure prophylaxis distribution, and initiating public health risk communications.
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From page 162... ...
Roberts and colleagues (2013) described a digital PCR diagnostic assay for ocular Chlamydia trachomatis; unlike other nucleic-acid amplification tests, it "requires no external or internal calibration yet delivers a highly accurate estimation of target load." Speaking about quantification, Morisset and colleagues (2013)
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From page 163... ...
However, assuming that Poisson statistics apply, 1/e = 37 percent of the time there will be no target in the PCR reaction, and one can, therefore, expect to "miss" the detection of this aerosolized bacteria at this aerosol concentration 37 percent of the time. By contrast, if there were, say, three separate PCR chambers, each with 10 percent overall throughput from collector to it, each with a different sequence signature from the bacterium, then the bacterium would miss all three reactions only 1/e3 = 5 percent of the time.
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From page 164... ...
A number of skilled laboratorians endorse the BioPlex® assay, based on multiplex PCR amplification and subsequent hybridization to a liquid array of bead types. A different group of skilled laboratorians endorses real-time PCR with Taqman® or Molecular Beacon® probes.
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From page 165... ...
Direct Sequencing of Probe Regions Direct sequencing of probe regions can be done via pyrosequencing, single-molecule sequencing, etc. Although next-generation sequencing continues to increase the power and affordability of sequencing a human's genome, how well do these increases enhance rapid, autonomous sequencing of pathogenic bacteria and viruses from complex environmental samples?
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From page 166... ...
using a surface-recognition capture, possibly with derivatized magnetic beads; Spore and cell lysis; Extraction and purification of nucleic acids; Possible enrichment and capture of nucleic-acid sequences that are signatures of virulence in human pathogens; Whatever amplification is necessary (perhaps priming off of the virulence regions) ; Fragmentation and size selection, as needed; Addition of bar codes, as needed; Ligation, as needed; Adjustment of buffers, washing, and separation, as needed for every step to produce the necessary sequencing library; Sequencing with each read being ≈ 400 bases; Maybe quantification of starting copy number; and Informatics analysis of sequence, including "identification" of strain and substrain variants (which is particularly tricky for ss DNA or RNA viruses)
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From page 167... ...
Also, the carryover of reads from prior sequencing runs could prevent simple adoption of an existing sequencing platform. What choices are there for robotic or microfluidic platforms to perform sample handling and sample preparation for a customized sequencing instrument?
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From page 168... ...
The Northrop Grumman system appears to have an easier path forward to expansion of the number of assays (the liquid array approach has a built-in capability to handle up to 100 target sequences) and runs internal positive and negative controls for every polymerase-amplification reaction.
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APPENDIX G 169 FIGURE G-3 M-BAND. FIGURE G-4 IntegenX.
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From page 170... ...
The author gratefully acknowledges discussions with Thomas Bunt, Shea Gardner, Staci Kane, Pejman Naraghi-Arani, David Rakestraw, Marilyn Ramsey, Tom Slezak, Mark Wagner, and Lewis Wogan of Lawrence Livermore National Laboratory; John Dzenitis and Phillip Belgrader of BioRad; M Allen Northup, formerly of Cepheid and MFSI; David Walt, Tufts University; Stevan Jovanovich, IntegenX; Kimothy Smith and Lyle Probst of PositiveID/MFSI; George Dizikes, Illinois Department of Public Health; Brent Chyna, State of Illinois and Astrix Technology Group, Inc.; Bernadette Johnson, Lincoln Labs; Cynthia Bruckner-Lea, Pacific Northwest National Laboratory; Michael Farrell, Centers for Disease Control and Prevention; and Darren Link, Raindance.
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2013. Detection of methicillin-resistant Staphylococcus aureus by a duplex droplet digital polymerase chain reaction.
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From page 172... ...
2013. Development and evaluation of a next generation digital PCR diagnostic assay for ocular Chlamydia trachomatis infections.
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