High throughput screening for spores and vegetative forms of pathogenic B. anthracis by an internally controlled real-time PCR assay with automated DNA preparation

  • Marcus Panning
  • Stefanie Kramme
  • Nadine Petersen
  • Christian Drosten
Original Investigation

Abstract

Human infections with Bacillus anthracis have become rare but in cases of intentional release, masses of samples would have to be expected. Current PCR assays for anthrax are appropriate for use in single cases, but they have not been formulated for high throughput screening. This article describes a high throughput real-time PCR for anthrax, including automated sample preparation without the need for pre-culturing of samples. The assay detects single copies of target gene. An internal control monitors the whole assay including sample preparation. The limit of detection in blood was 1,066 (95%CI, 741–1,739) copies/ml, corresponding to 4.4–32.3 organisms/ml. Using spore preparations, 20 colony-forming units (CFU) per sample could be detected reliably (0.8 CFU per PCR). The extraction procedures depleted viable spores from solution by factors of 10,000 (automated procedure) and >100,000 (conventional column procedure). One hundred and ten clinical and environmental specimens were retested, 50 of them sampled during a period of heightened anthrax awareness in 2001. A widely used assay yielded two false positive results (cross-reaction with B. cereus), while the new assay tested all samples negative. The internal control operated stable in all clinical samples. The assay is capable of testing for anthrax in the high throughput mode.

Keywords

Fluorescent Resonance Energy Transfer Bacillus Anthracis Viable Spore Sheep Blood Agar Intentional Release 

Notes

Acknowledgments

This work was supported by grants from the Bundesministerium für Gesundheit und Soziales (No. 325-4539-85/3) and the Bundesamt für Bevölkerungsschutz und Katastrophenhilfe (BBK-F-440-00-1). We thank Britta Liedigk for excellent technical assistance. We are grateful to Drs. Nattermann and Ellerbrok, Robert Koch Institute, Berlin, for the donation of B. anthracis strain Sterne.

References

  1. 1.
    Dixon TC, Meselson M, Guillemin J, Hanna PC (1999) Anthrax. N Engl J Med 341:815–826PubMedCrossRefGoogle Scholar
  2. 2.
    Jackson PJ, Hugh-Jones ME, Adair DM, Green G, Hill KK, Kuske CR, Grinberg LM, Abramova FA, Keim P (1998) 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 USA 95:1224–1229PubMedCrossRefGoogle Scholar
  3. 3.
    Jernigan JA, Stephens DS, Ashford DA, Omenaca C, Topiel MS, Galbraith M, Tapper M, Fisk TL, Zaki S, Popovic T, Meyer RF, Quinn CP, Harper SA, Fridkin SK, Sejvar JJ, Shepard CW, McConnell M, Guarner J, Shieh WJ, Malecki JM, Gerberding JL, Hughes JM, Perkins BA (2001) Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg Infect Dis 7:933–944PubMedCrossRefGoogle Scholar
  4. 4.
    Patra G, Williams LE, Qi Y, Rose S, Redkar R, Delvecchio VG (2002) Rapid genotyping of Bacillus anthracis strains by real-time polymerase chain reaction. Ann NY Acad Sci 969:106–111PubMedCrossRefGoogle Scholar
  5. 5.
    Qi Y, Patra G, Liang X, Williams LE, Rose S, Redkar RJ, DelVecchio VG (2001) Utilization of the rpoB gene as a specific chromosomal marker for real-time PCR detection of Bacillus anthracis. Appl Environ Microbiol 67:3720–3727PubMedCrossRefGoogle Scholar
  6. 6.
    Ryu C, Lee K, Yoo C, Seong WK, Oh HB (2003) Sensitive and rapid quantitative detection of anthrax spores isolated from soil samples by real-time PCR. Microbiol Immunol 47:693–699PubMedGoogle Scholar
  7. 7.
    Oggioni MR, Meacci F, Carattoli A, Ciervo A, Orru G, Cassone A, Pozzi G (2002) Protocol for real-time PCR identification of anthrax spores from nasal swabs after broth enrichment. J Clin Microbiol 40:3956–3963PubMedCrossRefGoogle Scholar
  8. 8.
    Hurtle W, Bode E, Kulesh DA, Kaplan RS, Garrison J, Bridge D, House M, Frye MS, Loveless B, Norwood D (2004) Detection of the Bacillus anthracis gyrA gene by using a minor groove binder probe. J Clin Microbiol 42:179–185PubMedCrossRefGoogle Scholar
  9. 9.
    Hoffmaster AR, Meyer RF, Bowen MD, Marston CK, Weyant RS, Thurman K, Messenger SL, Minor EE, Winchell JM, Rassmussen MV, Newton BR, Parker JT, Morrill WE, McKinney N, Barnett GA, Sejvar JJ, Jernigan JA, Perkins BA, Popovic T (2002) Evaluation and validation of a real-time polymerase chain reaction assay for rapid identification of Bacillus anthracis. Emerg Infect Dis 8:1178–1182PubMedGoogle Scholar
  10. 10.
    Ellerbrok H, Nattermann H, Ozel M, Beutin L, Appel B, Pauli G (2002) Rapid and sensitive identification of pathogenic and apathogenic Bacillus anthracis by real-time PCR. FEMS Microbiol Lett 214:51–59PubMedCrossRefGoogle Scholar
  11. 11.
    Drago L, Lombardi A, Vecchi ED, Gismondo MR (2002) Real-time PCR assay for rapid detection of Bacillus anthracis spores in clinical samples. J Clin Microbiol 40:4399PubMedCrossRefGoogle Scholar
  12. 12.
    Cockerill FR 3rd, Smith TF (2004) Response of the clinical microbiology laboratory to emerging (new) and reemerging infectious diseases. J Clin Microbiol 42:2359–2365PubMedCrossRefGoogle Scholar
  13. 13.
    Bode E, Hurtle W, Norwood D (2004) Real-time PCR assay for a unique chromosomal sequence of Bacillus anthracis. J Clin Microbiol 42:5825–5831PubMedCrossRefGoogle Scholar
  14. 14.
    Bell CA, Uhl JR, Hadfield TL, David JC, Meyer RF, Smith TF, Cockerill FR 3rd (2002) Detection of Bacillus anthracis DNA by LightCycler PCR. J Clin Microbiol 40:2897–2902PubMedCrossRefGoogle Scholar
  15. 15.
    Welkos SL (1991) Plasmid-associated virulence factors of non-toxigenic (pX01-) Bacillus anthracis. Microb Pathog 10:183–198PubMedCrossRefGoogle Scholar
  16. 16.
    Kiratisin P, Fukuda CD, Wong A, Stock F, Preuss JC, Ediger L, Brahmbhatt TN, Fischer SH, Fedorko DP, Witebsky FG, Gill VJ (2002) Large-scale screening of nasal swabs for Bacillus anthracis: descriptive summary and discussion of the National Institutes of Health’s experience. J Clin Microbiol 40:3012–3016PubMedCrossRefGoogle Scholar
  17. 17.
    La Scola B, Fournier PE, Raoult D (2003) Searching for Bacillus anthracis in suspect powders: a French experience. J Clin Microbiol 41:524; author reply 524–525Google Scholar
  18. 18.
    Coker PR, Smith KL, Fellows PF, Rybachuck G, Kousoulas KG, Hugh-Jones ME (2003) Bacillus anthracis virulence in Guinea pigs vaccinated with anthrax vaccine adsorbed is linked to plasmid quantities and clonality. J Clin Microbiol 41:1212–1218PubMedCrossRefGoogle Scholar
  19. 19.
    Shieh YS, Baric RS, Sobsey MD (1997) Detection of low levels of enteric viruses in metropolitan and airplane sewage. Appl Environ Microbiol 63:4401–4407PubMedGoogle Scholar
  20. 20.
    Maher N, Dillon HK, Vermund SH, Unnasch TR (2001) Magnetic bead capture eliminates PCR inhibitors in samples collected from the airborne environment, permitting detection of Pneumocystis carinii DNA. Appl Environ Microbiol 67:449–452PubMedCrossRefGoogle Scholar
  21. 21.
    Queiroz AP, Santos FM, Sassaroli A, Harsi CM, Monezi TA, Mehnert DU (2001) Electropositive filter membrane as an alternative for the elimination of PCR inhibitors from sewage and water samples. Appl Environ Microbiol 67:4614–4618PubMedCrossRefGoogle Scholar
  22. 22.
    Drosten C, Panning M, Kramme S (2003) Detection of Mycobacterium tuberculosis by real-time PCR using pan-mycobacterial primers and a pair of fluorescence resonance energy transfer probes specific for the M. tuberculosis complex. Clin Chem 49:1659–1661PubMedCrossRefGoogle Scholar
  23. 23.
    Drosten C, Weber M, Seifried E, Roth WK (2000) Evaluation of a new PCR assay with competitive internal control sequence for blood donor screening. Transfusion 40:718–724PubMedCrossRefGoogle Scholar
  24. 24.
    Roth WK, Weber M, Seifried E (1999) Feasibility and efficacy of routine PCR screening of blood donations for hepatitis C virus, hepatitis B virus, and HIV-1 in a blood-bank setting. Lancet 353:359–363PubMedCrossRefGoogle Scholar
  25. 25.
    Drosten C, Seifried E, Roth WK (2001) TaqMan 5′-nuclease human immunodeficiency virus type 1 PCR assay with phage-packaged competitive internal control for high-throughput blood donor screening. J Clin Microbiol 39:4302–4308PubMedCrossRefGoogle Scholar
  26. 26.
    Roth WK, Weber M, Buhr S, Drosten C, Weichert W, Sireis W, Hedges D, Seifried E (2002a) Yield of HCV and HIV-1 NAT after screening of 3.6 million blood donations in central Europe. Transfusion 42:862–868CrossRefGoogle Scholar
  27. 27.
    Roth WK, Weber M, Petersen D, Drosten C, Buhr S, Sireis W, Weichert W, Hedges D, Seifried E (2002b) NAT for HBV and anti-HBc testing increase blood safety. Transfusion 42:869–875CrossRefGoogle Scholar
  28. 28.
    Drosten C, Chiu LL, Panning M, Leong HN, Preiser W, Tam JS, Gunther S, Kramme S, Emmerich P, Ng WL, Schmitz H, Koay ES (2004) Evaluation of advanced reverse transcription-PCR assays and an alternative PCR target region for detection of severe acute respiratory syndrome-associated coronavirus. J Clin Microbiol 42:2043–2047PubMedCrossRefGoogle Scholar
  29. 29.
    Smieja M, Mahony JB, Goldsmith CH, Chong S, Petrich A, Chernesky M (2001) Replicate PCR testing and probit analysis for detection and quantitation of Chlamydia pneumoniae in clinical specimens. J Clin Microbiol 39:1796–1801PubMedCrossRefGoogle Scholar
  30. 30.
    Elnifro EM, Ashshi AM, Cooper RJ, Klapper PE (2000) Multiplex PCR: optimization and application in diagnostic virology. Clin Microbiol Rev 13:559–570PubMedCrossRefGoogle Scholar
  31. 31.
    Polz MF, Cavanaugh CM (1998) Bias in template-to-product ratios in multitemplate PCR. Appl Environ Microbiol 64:3724–3730PubMedGoogle Scholar
  32. 32.
    Rosenstraus M, Wang Z, Chang SY, DeBonville D, Spadoro JP (1998) An internal control for routine diagnostic PCR: design, properties, and effect on clinical performance. J Clin Microbiol 36:191–197PubMedGoogle Scholar
  33. 33.
    Willems M, Moshage H, Nevens F, Fevery J, Yap SH (1993) Plasma collected from heparinized blood is not suitable for HCV-RNA detection by conventional RT-PCR assay. J Virol Meth 42:127–130CrossRefGoogle Scholar
  34. 34.
    Pham DG, Madico GE, Quinn TC, Enzler MJ, Smith TF, Gaydos CA (1998) Use of lambda phage DNA as a hybrid internal control in a PCR-enzyme immunoassay to detect Chlamydia pneumoniae. J Clin Microbiol 36:1919–1922PubMedGoogle Scholar
  35. 35.
    Johanson J, Abravaya K, Caminiti W, Erickson D, Flanders R, Leckie G, Marshall E, Mullen C, Ohhashi Y, Perry R, Ricci J, Salituro J, Smith A, Tang N, Vi M, Robinson J (2001) A new ultrasensitive assay for quantitation of HIV-1 RNA in plasma. J Virol Meth 95:81–92CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2006

Authors and Affiliations

  • Marcus Panning
    • 1
  • Stefanie Kramme
    • 1
  • Nadine Petersen
    • 1
  • Christian Drosten
    • 1
  1. 1.Clinical Virology GroupBernhard Nocht Institute for Tropical MedicineHamburgGermany

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