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Artificial plasmid engineered to simulate multiple biological threat agents

Abstract

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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References

  1. Andersson SG et al (1998) The genome sequence of Rickettsia prowazekii and the origin of mitochondria. Nature 396(6707):133–140 Nov 12

  2. Bauernfeind A, Roller C, Meyer D, Jungwirth R, Schneider I (1998) Molecular procedure for rapid detection of Burkholderia mallei and Burkholderia pseudomallei. J Clin Microbiol 36(9):2737–2741

  3. Blattner FR et al (1997) The complete genome sequence of Escherichia coli K-12. Science. 277(5331):1453–1474

  4. Campbell J, Lowe J, Walz S, Ezzell J (1993) Rapid and specific identification of Yersinia pestis by using a nested polymerase chain reaction procedure. J Clin Microbiol 31(3):758–759

  5. Carl M, Hawkins R, Coulson N, Lowe J, Robertson DL, Nelson WM, Titball RW, Woody JN (1992) Detection of spores of Bacillus anthracis using the polymerase chain reaction. J Infect Dis 165(6):1145–1148

  6. Chain PS et al (2004) Insights into the evolution of Yersinia pestis through whole-genome comparison with Yersinia pseudotuberculosis. Proc Natl Acad Sci USA 101(38):13826–13831

  7. Chain PS et al (2005) Whole-genome analyses of speciation events in pathogenic Brucellae. Infect Immun 73(12):8353–8361

  8. Charrel RN, La Scola B, Raoult D (2004) Multi-pathogens sequence containing plasmids as positive controls for universal detection of potential agents of bioterrorism. BMC Microbiol 4:21

  9. Debeaumont C, Falconnet PA, Maurin M (2005) Real-time PCR for detection of Brucella spp. DNA in human serum samples. Eur J Clin Microbiol Infect Dis 24(12):842–845

  10. DelVecchio VG et al (2002) The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc Natl Acad Sci USA 99(1):443–448

  11. Deng W et al (2002) Genome sequence of Yersinia pestis KIM. J Bacteriol 184(16):4601–4611

  12. Dixon TC, Messelson M, Guillemin J, Hanna PC (1999) Anthrax: review article. N Engl J Med 341(11):815–826

  13. Espy MJ, Cockerill FR III, Meyer RF Bowen MD, Poland GA, Hadfield TL, Smith TF (2002) Detection of smallpox virus DNA by LightCycler PCR. J Clin Microbiol 40:1985–1988

  14. Fedele CG, Negredo A, Molero F, Sánchez-Seco MP, Tenorio A (2006) Use of internally controlled real-time genome amplification for detection of variola virus and other orthopoxviruses infecting humans. J Clin Microbiol 44(12):4464–4470

  15. Halling SM et al (2005) Completion of the genome sequence of Brucella abortus and comparison to the highly similar genomes of Brucella melitensis and Brucella suis. J Bacteriol 187(8):2715–2726

  16. Holden MT et al (2004) Genomic plasticity of the causative agent of melioidosis, Burkholderia pseudomallei. Proc Natl Acad Sci USA 01(39):14240–14245

  17. Inglis TJ, Sagripanti JL (2006) Environmental factors that affect the survival and persistence of Burkholderia pseudomallei. Appl Environ Microbiol 72(11):6865–6875

  18. Ivanova N et al (2003) Genome sequence of Bacillus cereus and comparative analysis with Bacillus anthracis. Nature 423(6935):87–91

  19. Kim HS et al (2005) Bacterial genome adaptation to niches: divergence of the potential virulence genes in three Burkholderia species of different survival strategies. BMC Genomics 6:174

  20. Larsson P et al (2005) The complete genome sequence of Francisella tularensis, the causative agent of tularemia. Nat Genet 37(2):153–159

  21. Lindler LE et al (1998) Complete DNA sequence and detailed analysis of the Yersinia pestis KIM5 plasmid encoding murine toxin and capsular antigen Infect. Immun 66(12):5731–5742

  22. Loïez C, Herwegh S, Wallet F, Armand S, Guinet F, Courcol RJ (2003) Detection of Yersinia pestis in sputum by real-time PCR. J Clin Microbiol 41(10):4873–4875

  23. Long GW, Oprandy JJ, Narayanan RB, Fortier AH, Porter KR, Nacy CA (1993) Detection of Francisella tularensis in blood by polymerase chain reaction. J Clin Microbiol 31(1):152–154

  24. Makino SI, Iinuma-Okada Y, Maruyama T, Ezaki T, Sasakawa C, Yoshikawa M (1993) Direct detection of Bacillus anthracis DNA in animals by polymerase chain reaction. J Clin Microbiol 31(3):547–551

  25. Makino K et al (1998) Complete nucleotide sequences of 93-kb and 3.3-kb plasmids of an enterohemorrhagic Escherichia coli O157:H7 derived from Sakai outbreak. DNA Res 5(1):1–9

  26. Massung RF et al (1993) Potential virulence determinants in terminal regions of variola smallpox virus genome. Nature 366(6457):748–751

  27. Matar GM, Khneisser IA, Abdelnoor AM (1996) Rapid laboratory confirmation of human Brucellosis by PCR analysis of a target sequence on the 31-kilodalton Brucella antigen DNA. J Clin Microbiol 34(2):477–478

  28. McLeod MP et al (2004) Complete genome sequence of Rickettsia typhi and comparison with sequences of other rickettsiae. J Bacteriol 186(17):5842–5855

  29. Nierman WC et al (2004) Structural flexibility in the Burkholderia mallei genome. Proc Natl Acad Sci USA 101(39):14246–14251

  30. Norkina OV, Kulichenko AN, Gintsburg AL, Tuchkov IV, Popov YuA, Aksenov MU, Drosdov IG (1994) Development of a diagnostic test for Yersinia pestis by the polymerase chain reaction. J Appl Bacteriol 76(3):240–245

  31. O’Connell KP, Bucher JR, Anderson PE, Cao CJ, Khan AS, Gostomski MV, Valdes JJ (2006) Real-time fluorogenic reverse transcription-PCR assays for detection of bacteriophage MS2. Appl Environ Microbiol 72(1):478–483

  32. Ogata H et al (2001) Mechanisms of evolution in Rickettsia conorii and R. prowazekii. Science 293(5537):2093–2098

  33. Ogata H et al (2005) The genome sequence of Rickettsia felis identifies the first putative conjugative plasmid in an obligate intracellular parasite. PLoS Biol 3(8):e248 Aug

  34. Okinaka R et al (1999) Sequence, assembly and analysis of pX01 and pX02. J Appl Microbiol 87(2):261–262

  35. Okinaka RT et al (1999–2) Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J. Bacteriol 181(20):6509–6515

  36. Parkhill J et al (2001) Genome sequence of Yersinia pestis, the causative agent of plague. Nature 413(6855):523–527

  37. Paulsen IT (2002) The Brucella suis genome reveals fundamental similarities between animal and plant pathogens and symbionts. Proc Natl Acad Sci U.S.A 99(20):13148–13153

  38. Paulsen IT et al (2003) complete genome sequence of the Q-fever pathogen Coxiella burnetii. Proc Natl Acad Sci USA 100(9):5455–5460 Epub 2003 Apr 18

  39. Perna NT et al (2001) Genome sequence of enterohaemorrhagic Escherichia coli O157:H7. Nature 409(6819):529–533

  40. Ramisse V, Patra G, Garrigue H, Guesdon JL, Mock M (1996) Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA. FEMS Microbiol Lett 145(1):9–16

  41. Rasko DA et al (2004) The genome sequence of Bacillus cereus ATCC 10987 reveals metabolic adaptations and a large plasmid related to Bacillus anthracis pXO1. Nucleic Acids Res 32(3):977–988

  42. Read TD et al (2003) The genome sequence of Bacillus anthracis Ames and comparison to closely related bacteria. Nature 423(6935):81–86

  43. Sharma VK, Dean-Nystrom EA (2003) Detection of enterohemorrhagic Escherichia coli O157:H7 by using a multiplex real-time PCR assay for genes encoding intimin and Shiga toxins. Vet Microbiol. 93(3):247–260

  44. Shchelkunov SN et al (1994) Analysis of the nucleotide sequence of 53 kbp from the right terminus of the genome of variola major virus strain India-1967. Virus Res 34(3):207–236

  45. Song Y et al (2004) Complete genome sequence of Yersinia pestis strain 91001, an isolate avirulent to humans DNA. Res 11(3):179–197

  46. Stein A, Raoult D (1992) Detection of Coxiella burnetti by DNA amplification using polymerase chain reaction. J Clin Microbiol 30(9):2462–2466

  47. Tomaso H, Reisinger EC, Al Dahouk S, Frangoulidis D, Rakin A, Landt O, Neubauer H (2003) Rapid detection of Yersinia pestis with multiplex real-time PCR assays using fluorescent hybridisation probes. FEMS Immunol Med Microbiol 38(2):117–126

  48. Tzianabos T, Anderson BE, McDade JE (1989) Detection of Rickettsia rickettsii DNA in clinical specimens by using polymerase chain reaction technology. J Clin Microbiol 27(12):2866–2868

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Acknowledgment

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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Keywords

  • 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