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Artificial plasmid engineered to simulate multiple biological threat agents


The objective of this study was to develop a non-virulent simulant to replace several virulent organisms during the development of detection and identification methods for biological threat agents. We identified and selected specific genes to detect Yersinia pestis, Francisella tularensis, Burkholderia mallei, Burkholderia pseudomallei, Rickettsia sp., Coxiella burnetii, Brucella sp., enterohemorrhagic Escherichia coli O157:H7, Bacillus anthracis, and variola (smallpox) virus. We then designed and engineered a non-infectious simulant that included the nucleic-acid signature of each microorganism in a single chimerical molecule. Here, we reported an approach that by direct (de novo) chemical synthesis permitted the production of a single chimerical construct 2,040bp long that included the nucleic-acid signature of the bacterial and viral biological threat agents listed above without requiring access to these agents. Sequences corresponding to each one of the biological agents in the synthetic simulant were amplified by PCR, resulting in amplicons of the expected length, of similar intensity, and without any detectable unspecific products. The novel simulant described here could reduce the need for infectious agents in the development of detection and diagnostic methods and should also be useful as a non-virulent positive control in nucleic-acid-based tests against biological threat agents.

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This work was supported by the In-House Laboratory Independent Research (ILIR) funds from the Research and Technology Directorate, Edgewood Chemical Biological Center, Research Development and Engineering Command, US Army.

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Correspondence to Jose-Luis Sagripanti.

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Carrera, M., Sagripanti, J. Artificial plasmid engineered to simulate multiple biological threat agents. Appl Microbiol Biotechnol 81, 1129–1139 (2009). https://doi.org/10.1007/s00253-008-1715-8

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  • Threat agents
  • Bioinformatics
  • PCR
  • Multiplex
  • Bacillus anthracis
  • Yersinia pestis
  • Coxiellla burnetii
  • Brucella
  • Francisella tularensis
  • Enterohemorrhagic E. coli O157:H7
  • Burkholderia mallei
  • Burkholderia pseudomallei
  • Variola
  • Smallpox