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Volume 8, Number 10—October 2002

Evaluation and Validation of a Real-Time Polymerase Chain Reaction Assay for Rapid Identification of Bacillus anthracis

Alex R. Hoffmaster, Richard F. Meyer, Michael P. Bowen, Chung K. Marston, Robbin S. Weyant, Kathy Thurman, Sharon L. Messenger, Erin E. Minor, Jonas M. Winchell, Max V. Rasmussen, Bruce R. Newton, J. Todd Parker, William E. Morrill, Nancy McKinney, Gwen A. Barnett, James J. Sejvar, John A. Jernigan, Bradley A. Perkins, and Tanja Popovic
Author affiliations: Centers for Disease Control and Prevention, Atlanta, Georgia, USA;

Main Article


Origin, designations, and results of real-time polymerase chain reaction assay for Bacillus anthracis strainsa

No. positive/total
B. anthracis No. analyzed Temporal range and geographic origin MLVA genotypes representeda Ba1b Ba2b Ba3b
Human isolates 30 1943–1996 Africa, Asia, Australia, Europe, North America 3, 4, 22, 23, 28, 32, 34, 35, 36, 37, 41, 43, 44, 45, 50, 66, 68 30/30 30/30 30/30
Animal isolates 29 Africa, Asia, Australia, Europe, North America, South America 3, 10, 20, 26, 29, 30, 35, 38, 40, 45, 48, 49, 51, 55, 57, 78, 80, 81, 84, 85, 87, 89 29/29 29/29 29/29
Other isolates 16 1950–1993
 Africa, Asia, Europe, N. America 13, 14, 21, 24, 47, 62, 69, 73, 77, 79, 82 16/16 16/16 16/16
Outbreak isolates 317 2001
 U.S. outbreak 62 317/317 317/317 317/317
pXO1 cured 5 1956–1974
 North America 5/5 0/5 5/5
pXO2 cured 1 Africa 0/1 1/1 1/1

aMLVA, multiple-locus variable-number tandem repeat analysis as described by Keim et al. (15).
bBa1, Ba2, and Ba3 primer/probe sets as described in Materials and Methods.

Main Article

  1. Makino  SI, Cheun  HI, Watarai  M, Uchida  I, Takeshi  K. Detection of anthrax spores from the air by real-time PCR. Lett Appl Microbiol. 2001;33:23740. DOIPubMedGoogle Scholar
  2. Makino  SI, Iinuma-Okada  Y, Maruyama  T, Ezaki  T, Sasakawa  C, Yoshikawa  M. Direct detection of Bacillus anthracis DNA in animals by polymerase chain reaction. J Clin Microbiol. 1993;31:54751.PubMedGoogle Scholar
  3. Lee  MA, Brightwell  G, Leslie  D, Bird  H, Hamilton  A. Fluorescent detection techniques for real-time multiplex strand specific detection of Bacillus anthracis using rapid PCR. J Appl Microbiol. 1999;87:21823. DOIPubMedGoogle Scholar
  4. Turnbull  PC, Hutson  RA, Ward  MJ, Jones  MN, Quinn  CP, Finnie  NJ, Bacillus anthracis but not always anthrax. J Appl Bacteriol. 1992;72:218.PubMedGoogle Scholar
  5. Okinaka  RT, Cloud  K, Hampton  O, Hoffmaster  AR, Hill  KK, Keim  P, Sequence and organization of pXO1, the large Bacillus anthracis plasmid harboring the anthrax toxin genes. J Bacteriol. 1999;181:650915.PubMedGoogle Scholar
  6. Okinaka  RT, Cloud  K, Hampton  O, Hoffmaster  AR, Hill  KK, Keim  P, Sequence, assembly and analysis of pXO1 and pXO2. J Appl Microbiol. 1999;87:2612. DOIPubMedGoogle Scholar
  7. Green  BD, Battisti  L, Koehler  TM, Thorne  CB, Ivins  BE. Demonstration of a capsule plasmid in Bacillus anthracis. Infect Immun. 1985;49:2917.PubMedGoogle Scholar
  8. Pannucci  J, Okinaka  RT, Sabin  R, Kuske  CR. Bacillus anthracis pXO1 plasmid sequence conservation among closely related bacterial species. J Bacteriol. 2002;184:13441. DOIPubMedGoogle Scholar
  9. Ramisse  V, Patra  G, Garrigue  H, Guesdon  GL, Mock  M. Identification and characterization of Bacillus anthracis by multiplex PCR analysis of sequences on plasmids pXO1 and pXO2 and chromosomal DNA. FEMS Microbiol Lett. 1996;145:916. DOIPubMedGoogle Scholar
  10. Ramisse  V, Patra  G, Vaissaire  J, Mock  M. The Ba813 chromosomal DNA sequence effectively traces the whole Bacillus anthracis community. J Appl Microbiol. 1999;87:2248. DOIPubMedGoogle Scholar
  11. Patra  G, Vaissaire  J, Weber-Levy  M, Le Doujet  C, Mock  M. Molecular characterization of Bacillus strains involved in outbreaks of anthrax in France in 1997. J Clin Microbiol. 1998;36:34124.PubMedGoogle Scholar
  12. Jackson  PJ, Hugh-Jones  ME, Adair  DM, Green  G, Hill  KK, Kuske  CR, PCR analysis of tissue samples from the 1979 Sverdlovsk anthrax victims: the presence of multiple Bacillus anthracis strains in different victims. Proc Natl Acad Sci U S A. 1998;95:12249. DOIPubMedGoogle Scholar
  13. Qi  Y, Patra  G, Liang  X, Williams  LE, Rose  S, Redkar  RJ, Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis. Appl Environ Microbiol. 2001;67:37207. DOIPubMedGoogle Scholar
  14. Khan  AS, Morse  S, Lillibridge  SR. Public-health preparedness for biological terrorism in the USA. Lancet. 2000;356:117982. DOIPubMedGoogle Scholar
  15. Keim  P, Price  LB, Klevytska  AM, Smith  KL, Schupp  JM, Okinaka  R, Multiple-locus variable-number tandem repeat analysis reveals genetic relationships within Bacillus anthracis. J Bacteriol. 2000;182:292836. DOIPubMedGoogle Scholar
  16. Plotkin  SA, Brachman  PS, Utell  M, Bumford  FH, Atchison  MM. An epidemic of inhalation anthrax, the first in the twentieth century. Am J Med. 1960;29:9921001. DOIPubMedGoogle Scholar
  17. Hoffmaster  AR, Fitzgerald  CC, Ribot  E, Mayer  LW, Popovic  T. Importance of molecular subtyping of B. anthracis during the recent multi-state bioterrorism-associated anthrax outbreak in the United States. Emerg Infect Dis. 2002.
  18. Logan  NA, Turnbull  PC. Bacillus and recently derived genera. In: Murray PR, editor. Manual of clinical microbiology. Washington (DC): ASM Press; 2001. p. 357–69.
  19. Helgason  E, Okstad  OA, Caugant  DA, Johansen  HA, Fouet  A, Mock  M, Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—one species on the basis of genetic evidence. Appl Environ Microbiol. 2000;66:262730. DOIPubMedGoogle Scholar
  20. Ash  C, Farrow  JA, Dorsch  M, Stackebrandt  E, Collins  MD. Comparative analysis of Bacillus anthracis, Bacillus cereus, and related species on the basis of reverse transcriptase sequencing of 16S rRNA. Int J Syst Bacteriol. 1991;41:3436.PubMedGoogle Scholar
  21. Kaneko  T, Nozaki  R, Aizawa  K. Deoxyribonucleic acid relatedness between Bacillus anthracis, Bacillus cereus and Bacillus thuringiensis. Microbiol Immunol. 1978;22:63941.PubMedGoogle Scholar
  22. Ticknor  LO, Kolsto  AB, Hill  KK, Keim  P, Laker  MT, Tonks  M, Fluorescent amplified fragment length polymorphism analysis of Norwegian Bacillus cereus and Bacillus thuringiensis soil isolates. Appl Environ Microbiol. 2001;67:486373. DOIPubMedGoogle Scholar
  23. Cockerill  FR, Smith  TF. Rapid-cycle real-time PCR: a revolution for clinical microbiology. ASM News. 2002;68:7783.
  24. Livak  KJ, Flood  SJ, Marmaro  J, Giusti  W, Deetz  K. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl. 1995;4:35762.PubMedGoogle Scholar

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