Sensor Systems for Biological Agent Attacks Protecting Buildings and Military Bases (2005) / Chapter Skim
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6 Nucleic Acid Sequence-Based Identification for Detect-to-Warn Applications
6 Nucleic Acid Sequence-Based Identification for Detect-to-Warn Applications
Pages 84-104
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From page 84... ...
However, nucleic acid-based assays offer a relatively faster response bme combined with high sensitivity and specificity, and this has catapulted them to the forefront for laboratory analysis and, in some cases, field analysis. The sequence of nucleic acids has been successfully used in laboratory settings to identify both nonpathogenic' and pathogenic bacteria.2 Similar studies of microbial diversity have been performed using ribosomal RNA methods.3 It is the view of this committee that sequence-based ID using nucleic acid assays will play a critical role in any defensive architecture against biological agent attack, to conf mm whether or not an attack has occurred and if it has, what types of biological agents were used.4 This confimmatory role is essential even if the response time of the assay is relatively slow—on the order of 1~30 minutes.
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From page 85... ...
The same considerations apply to the use of multistage precollection fractionators that could provide concentration faders exceeding 105; improved overall perfommance is likely to come at the expense of more frequent maintenance. SAMPLE PREPARATION Nucleic acid amplification and analysis methods are sensitive to contamination by inhibitors, which can often accompany samples that are collected from the open environment.
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From page 86... ...
Boom has been implemented using microfabricated silica surfaces within micnafluidic systems.9 The committee is not aware, however, of any studies on the rates or kinetics involved in the use of such micnafluidic silica surfaces for the overall process of trapping, washing, and releasing nucleic acids from Iysed bacteria. Using the chemistry of Boom, Ness and Belgrader have demonstrated the polymerase chain reaction (PCR)
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From page 87... ...
There are no data regarding the percent of DNA or RNA that will be released by this process, but a reasonable guess might be that roughly 30 percent of the nucleic acids from spores or vegetative bacteria could be released within a sample volume of 100 microliters or less (see Box 6.1)
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From page 88... ...
With a single copy of the genome per bacterium, there are 600 target sequences. · For 20 seconds, use a pressure-driven or electrophoretic or other voltage-driven transport mechanism to move the DNA onto a sample preparation surface.
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From page 89... ...
are synthesized in known sequences and attached to spots in the array in a known and predetemmined way. Detection of the spots that contain hybridized target DNA after the hybridization event allows one to infer the sequence of DNA in the unknown target.
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From page 90... ...
:1549-1559. Although a well-developed PCR assay is capable of detecting a single target DNA sequence, one cannot reliably detect unknowns at such low levels, even when inhibitors have been removed This can be understood simply by Poisson's distribution, assuming a random sampling process if the average number of target DNA sequences in a sample is 1 (the product of concentration bmrssample volume = 1)
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From page 91... ...
, for each copy of target DNA sequence that was present when the reaction process began, the reaction will have manufactured (2N-1) copies.
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From page 92... ...
, real-bme PCR has provided increased sensitivity, specificity, and rapidity of the assay.33 When the samples are well characterized, it is possible both to detect and to quantify the number of starting copies of target DNA in the PCR. When PCR is used without fluorogenic probes, the product is typically detected using gel or capillary electrophoresis or via hybridization against an array or microarray of probes (see discussion below on microarrays and HySeq patents and Affymax patents)
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From page 93... ...
that contain many repeats of the sequence in the circular template.39 The small circular template can be used as a probe to bind to and thereby identify unknown target DNA. After the RCA is completed, the target DNA ends up being attached to a long strand of cDNA.
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From page 94... ...
This recognition sequence is nicked in each cycle and a fragment of an appropriate polymerase binds to this site, replicates the complementary 3'-5' target strand, and the enzyme displaces the original 5' 3'strand, freeing it to diffuse away in solution (see Figure 6.6) .42 After an initial heating step to denature the original target DNA, this process proceeds isothermally without heating cycles and ,/ can produce 1 o'2 copies of a single target f DNA sequence in 30 minutes at 370C 43 An in situ hybridization version of the technique has been used to detect as few as one gene copy of human immunodeficiency vinus (HIV)
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From page 95... ...
, also known as the ligase chain reaction (LCR) .48 This method, mentioned briefly above, is similar to PCR in its need for themmal cycling but uses two oligonucleobde probes that bind to adjacent sites on the target DNA at the lower temperature of the themmal cycle.
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From page 96... ...
Target DNA strands that are washed onto the arTay and allowed to settle will hybridize to oligos having an exactly complementary sequence but will only weakly bind to oligos that do not have an exact match if the chemistry of the solution is properly adjusted. The hybridized target DNA is usually labeled in some manner such as with a fluorescent dye molecule so that the pattern of hybridization across the array can be detected.
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From page 97... ...
One of the most important drawbacks is that the technology typically uses PCR to amplify the amount of target DNA before applying it to the array to maximize the signal. These factors all combine to make the conventional DNA microarray technology unsuitable for detect-to-wam applications.
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From page 98... ...
· Use 10 seconds to collect a sample from the air into an aqueous solution, using a collector with a two-stage, Recollection fractionator possessing an overall capture efficiency of 50 percent and a collection rate of 90 liters of air per minute into 50 microliters of aqueous volume; 100 spores per liter of air would produce 750 spores in the 50 microliters of solution.b · Use 5 seconds to extract 10 microliters of the solution for analysis, archiving the remaining 40 microliters and/or providing it for parallel assays.C · Ultrasonicate for 10 seconds to get access to the nucleic acids inside the spores. Due to the short bme, assume access is gained to only 33 percent of the ribosomal RNA.
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From page 99... ...
Also, although the committee has seen data on label-free hybridization of nucleic acids using a swelling of a holographic-grating film (Chnstopher Lowe, University of Cambridge, presentation to the Group 11: Sequence-Based Assays That Do Not Use Amplification Techniques Although there have been musings about directly reading the sequence of DNA by using a mass spectrometer or a scanning-probe microscope or a nanometer orifice, the techniques that are used today to determine the sequence of RNA or DNA are based upon hybridizabon.59 These techniques include sequencing by hybridization, such as array-based techniques.59 Multiple signal-transduction mechanisms are also possible, including signal amplification, which the committee views as different from sequence amplification. One example of a signal amplification technique is the use of a label that chemiluminesces or that can undergo repeated electrochemical oxidation-reducton reactions.
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From page 100... ...
are molecules that exhibit Relatively little fluorescence when unbound to target DNA but fluoresce when bound to their complementary sequence rut Molecular beacons could be attractive for the detect-to-warn application if means could be found to bring them together with the DNA or RNA in cells quickly, perhaps using electric-field effects or other methods to ovencome the time lag due to diffusion as described above. Trace-level detection could be enhanced for the molecular beacons via development of quenchable Stokes-shift dyes (similar to Cy3 or Cy5)
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From page 101... ...
When a short single strand of target DNA hybridizes to the oligo, however, a rigid doublestranded section of DNA is fommed that pulls the fluorophore from the surface of the nanoparUde, allowing it to emit light again. Nie and coworkers have also shown that Nanometer fluorescent nanoparticles can be conjugated to DNA-binding proteins such as the restriction enzyme EcoRI, bound to specific sites on target DNA strands, and imaged once bound.
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From page 102... ...
The committee notes several promising developments that could help to reduce the response times of sequence-based detector/idenbfier systems. The basic process that leads to spore disnuption and cell Iysis during ultrasonication with beads is becoming better understood—beads may not be necessary at all; nano- or microbubbles may be all that are needed—and it may be possible to design a sampleprocessing front end to extract ribosomal RNA rapidly.
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From page 103... ...
Due to flhe much greater length of flhe bacterial genomic DNA, the DNA affords better identification of both species and strain of a bacterium than does the rRNA, and an rRNA assay alone may be more appropriate as a rapid screening tool than as a tool for the precise identification of the bioagent.73 However, flhe committee suggests a notional detector flhat uses a hybridization assay with a sequence of rRNA as its identifier because flhe presence of 10,000 copies of the rRNA in each bacterium may enable detection without resorting to the more time-consuming step of amplification of the nucleic acid. FINDINGS AND RECOMMENDATIONS Detection and identification of organisms by assays based on nucleic acid sequencing are accepted worldwide as offering the greatest infommation content and sensitivity of any single technology.
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From page 104... ...
Finding 6~4: Although a detect-to-warn system has its highest impact if it can initiate responses within approximately 1 minute of an attack, technologies that provide confimmabon of the attack and identify the organisms involved will serve a vital function in the overall defensive architecture, even if their response times are several minutes. Recommendation 6-4: R&D should be conducted to develop an integrated, fully automated PCR system, including sample collection, preparation, and analysis, initially with a 1 5-minute or so overall confimmabon time and later with a 5-minute or so confirmation time.
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