Pathogen detection using the Luminex multi-analyte system
Item
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Title
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Pathogen detection using the Luminex multi-analyte system
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Identifier
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d_2009_2013:1954938c21b1:11139
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identifier
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11515
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Creator
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Schweighardt, Andrew J.,
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Contributor
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Margaret M. Wallace
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Date
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2012
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Language
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English
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Publisher
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City University of New York.
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Subject
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Molecular biology | Luminex | pathogens
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Abstract
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The causative agents of anthrax, botulism, tularemia, and plague are so destructive that they could be used maliciously by bioterrorists. Microscopical and biochemical methods of identifying pathogenic bacteria are often inaccurate and time-consuming. Genetics-based alternatives surpass traditional detection methods in terms of speed and accuracy, but often lack the capacity for multiplexing.;Luminex technology embodies all the advantages of DNA-based identification while still offering impressive multiplexing capabilities. Luminex is a liquid array platform that relies on specially labeled microscopic beads to which unique probes are covalently attached. Hybridization of the probes to the unknown target sequences is detected and quantified by the instrument.;Four core pathogenic bacterial species were selected: Bacillus anthracis, Clostridium botulinum, Francisella tularensis subsp. tularensis , and Yersinia pestis. Probe sequences intended for confirmatory identification of the four core pathogens were located within genes related to the toxicity of each bacterium. Additional probe sequences within the 23S ribosomal RNA gene rrl were selected for presumptive identification of the four core pathogens, or a relative belonging to the same genus.;Primer3 and AutoDimer software programs were used during probe and target DNA design to specify oligo parameters and minimize cross-reactivity. The specificity of all oligos was verified using whole genome searches conducted on the NCBI BLAST database. Target sequences were amplified by PCR and quantified using the Agilent 2100 Bioanalyzer. Primer specificity was confirmed by the detection of target band(s) of the requisite length.;Specificity and sensitivity were examined by introducing various increments of each target sequence to all probes. A reasonable degree of specificity was observed, although some probes were prone to false positives. The LLDs for the probes ranged from 0.1 to 10 ng. Sensitivity was improved by using lambda exonuclease to enzymatically digest the non-complementary PCR strand.;Thirty-three binary, ternary, and quaternary mixtures were examined in which all components were present in a 1:1 ratio, with an 80% success rate for identifying all components. Four binary mixtures were subjected to further study in which the two components were combined in various ratios. Both components were detected in 100% of the twenty-eight mixtures analyzed, even when the minor component was overshadowed by a ten-fold excess of the major component. False positives typically had an attenuated fluorescent response that made them easily distinguishable from a genuine positive result.;Overall, the Luminex platform is a robust method of identifying pathogenic microorganisms. This technique has a superior level of specificity and sensitivity, provided that each assay has its underpinnings in a thorough survey of all pertinent bioinformatics data. Continued research with the Luminex technology is important both for validating the utility of this technique and for satisfying legal admissibility standards.
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Type
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dissertation
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Source
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2009_2013.csv
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degree
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Ph.D.
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Program
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Criminal Justice
