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Chapter 33 : Detection of Agents Associated with Bioterrorism

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Detection of Agents Associated with Bioterrorism, Page 1 of 2

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Abstract:

In this current age of continued threat of large-scale infectious disease outbreaks, whether intentional or natural, the importance of quickly identifying and characterizing the causes of the outbreaks has never been greater. This chapter provides a brief look at the issues and needs related to biothreat detection and the systems and technology developed, or under development, to address them. While anthrax is the most visible and perhaps most likely biothreat disease, others are predicted to have high biothreat potential as well. Real-time PCR has become one of the most widely used tools in diagnostics and biothreat pathogen detection. The focus of confirmatory testing for select agents is real-time PCR technology. BioWatch is an environmental surveillance system set up to continuously monitor air samples for biothreat agents, with the purpose of providing an early-warning system for public policy, safety, and health officials at the local level. There have been a number of positive results reported in the news media, although these are generally classified as background noise from naturally occurring pathogens. There are multiple approaches to next-generation sequencing, but two of the most promising (SOLiD and Illumina GA) are based on very short read lengths that are still suitable for whole-genome genotyping.

Citation: Keim P, Waddell V, Engelthaler D. 2011. Detection of Agents Associated with Bioterrorism, p 525-534. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch33

Key Concept Ranking

Severe Acute Respiratory Syndrome
0.45637584
Real-Time PCR
0.45281032
Culture Methods
0.43612322
Microbiological Methods
0.4021918
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Figures

Image of FIGURE 1
FIGURE 1

Standard sensitivity curve for dual-probe real-time PCR of DNA. Highly specific PCR assays can be developed using two TaqMan probes that bind to the same region of the PCR amplicon, but to slightly different internal amplicon DNA sequences. This particular example uses single nucleotide polymorphisms to distinguish a particular strain (e.g., the Ames strain) from all other strains ( ). The two different fluorescently labeled probes compete for binding to the DNA as the amplicon increases in concentration with each cycle. The DNA polymerase used in PCR preferentially cleaves the probe that binds the best to amplicon and releases that probe’s fluorescent label. In the probes’ competition there is rarely a complete winner such that the ratio of the two probes is important for the analysis. (A) Real-time fluorescence is plotted ( axis) as the PCR amplification progresses through multiple cycles ( axis) for quantitative analysis. The use of higher concentrations of initial DNA template results in more rapid amplification of the product. In this example, 1 ng is the highest amount of DNA added and 10 fg the lowest. The midpoint on the amplification curve can be used in conjunction with a standard curve to determine an accurate estimate of the amount of a target in a clinical or environmental sample. The probe that matches the amplicon sequence exactly increases dramatically (labeled lines), while the mismatched probe is increased only slightly (unlabeled lines near the axis). (B) Endpoint analysis of real-time PCR is based upon the relative fluorescence of the two probes. This analysis is used for qualitative determinations of an unknown sample with the position of each sample used for identification. The dots indicate the fluorescence of the sample with its proximity to each axis acting as a diagnostic (note the distinct clusters).

Citation: Keim P, Waddell V, Engelthaler D. 2011. Detection of Agents Associated with Bioterrorism, p 525-534. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch33
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Image of FIGURE 2
FIGURE 2

The integrated LRN structure ( ). (Art work courtesy of Talima Pearson.)

Citation: Keim P, Waddell V, Engelthaler D. 2011. Detection of Agents Associated with Bioterrorism, p 525-534. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch33
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Image of FIGURE 3
FIGURE 3

A handheld immunoassay (Neogen [http://www.neogen.com]). These are lateral flow devices called “Quick Sticks” where the sample is added in the circular opening immediately below the “T.” The sample flows by capillary action across a line of capture antibodies specific for the target antigen (e.g., a spore-associated moiety) or for a control antigen (upper line). The two-line staining pattern is indicative for presence of the target and whether the assays are working correctly. Four cases are illustrated: 1, invalid result; 2, positive + control; 3, positive control; 4, negative. (Photo courtesy of Arizona Department of Health Services.)

Citation: Keim P, Waddell V, Engelthaler D. 2011. Detection of Agents Associated with Bioterrorism, p 525-534. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch33
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References

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Tables

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TABLE 1

Microbiological methods used for biothreat agents

Citation: Keim P, Waddell V, Engelthaler D. 2011. Detection of Agents Associated with Bioterrorism, p 525-534. In Persing D, Tenover F, Tang Y, Nolte F, Hayden R, van Belkum A (ed), Molecular Microbiology. ASM Press, Washington, DC. doi: 10.1128/9781555816834.ch33

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