Skip to main content

Advertisement

SpringerLink
Log in

Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples

  • Methods and Protocols
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

A real-time polymerase chain reaction (PCR) assay was developed for rapid identification of Bacillus anthracis in environmental samples. These samples often harbor Bacillus cereus bacteria closely related to B. anthracis, which may hinder its specific identification by resulting in false positive signals. The assay consists of two duplex real-time PCR: the first PCR allows amplification of a sequence specific of the B. cereus group (B. anthracis, B. cereus, Bacillus thuringiensis, Bacillus weihenstephanensis, Bacillus pseudomycoides, and Bacillus mycoides) within the phosphoenolpyruvate/sugar phosphotransferase system I gene and a B. anthracis specific single nucleotide polymorphism within the adenylosuccinate synthetase gene. The second real-time PCR assay targets the lethal factor gene from virulence plasmid pXO1 and the capsule synthesis gene from virulence plasmid pXO2. Specificity of the assay is enhanced by the use of minor groove binding probes and/or locked nucleic acids probes. The assay was validated on 304 bacterial strains including 37 B. anthracis, 67 B. cereus group, 54 strains of non-cereus group Bacillus, and 146 Gram-positive and Gram-negative bacteria strains. The assay was performed on various environmental samples spiked with B. anthracis or B. cereus spores. The assay allowed an accurate identification of B. anthracis in environmental samples. This study provides a rapid and reliable method for improving rapid identification of B. anthracis in field operational conditions.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

References

  • Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402

    Article  CAS  Google Scholar 

  • Antwerpen MH, Zimmermann P, Bewley K, Frangoulidis D, Meyer H (2008) Real-time PCR system targeting a chromosomal marker specific for Bacillus anthracis. Mol Cell Probes 22:313–315

    Article  CAS  Google Scholar 

  • Beyer W, Glockner P, Otto J, Bohm R (1995) A nested PCR method for the detection of Bacillus anthracis in environmental samples collected from former tannery sites. Microbiol Res 150:179–186

    CAS  Google Scholar 

  • Cheun HI, Makino SI, Watarai M, Erdenebaatar J, Kawamoto K, Uchida I (2003) Rapid and effective detection of anthrax spores in soil by PCR. J Appl Microbiol 95:728–733

    Article  CAS  Google Scholar 

  • Dixon TC, Meselson M, Guillemin J, Hanna PC (1999) Anthrax. N Engl J Med 341:815–826

    Article  CAS  Google Scholar 

  • Easterday WR, Van Ert MN, Zanecki S, Keim P (2005) Specific detection of Bacillus anthracis using a TaqMan mismatch amplification mutation assay. Biotechniques 38:731–735

    Article  CAS  Google Scholar 

  • 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 214:51–59

    Article  CAS  Google Scholar 

  • Gonzy-Tréboul G, Steinmetz M (1987) Phosphoenolpyruvate:sugar phosphotransferase system of Bacillus subtilis: cloning of the region containing the ptsH and ptsI genes and evidence for a crr-like gene. J Bacteriol 169:2287–2290

    Google Scholar 

  • Hadjinicolaou AV, Demetriou VL, Hezka J, Beyer W, Hadfield TL, Kostrikis LG (2009) Molecular beacons and multi-allelic real-time PCR for detection of and discrimination between virulent Bacillus anthracis and other Bacillus isolates. J Microbiol Methods 78:45–53

    Article  CAS  Google Scholar 

  • Harling R, Twisselmann B, Asgari-Jirhandeh N, Morgan D, Lightfoot N, Reacher M, Nicoll A, Deliberate Release Teams (2001) Deliberate releases of biological agents: initial lessons for Europe from events in the United States. Euro Surveill 6:166–167

    CAS  Google Scholar 

  • Helgason E, Okstad OA, Caugant DA, Johansen HA, Fouet A, Mock M, Hegna I, Kolsto AB (2000) Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis—one species on the basis of genetic evidence. Appl Environ Microbiol 66:2627–2630

    Article  CAS  Google Scholar 

  • 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 8:1178–1182

    CAS  Google Scholar 

  • Hoffmaster AR, Ravel J, Rasko DA, Chapman GD, Chute MD, Marston CK, De BK, Sacchi CT, Fitzgerald C, Mayer LW, Maiden MC, Priest FG, Barker M, Jiang L, Cer RZ, Rilstone J, Peterson SN, Weyant RS, Galloway DR, Read TD, Popovic T, Fraser CM (2004) Identification of anthrax toxin genes in a Bacillus cereus associated with an illness resembling inhalation anthrax. Proc Natl Acad Sci USA 101:8449–8454

    Article  CAS  Google Scholar 

  • Hoffmaster KK Hill, Gee JE, Marston CK, De BK, Popovic T, Sue D, Wilkins PP, Avashia SB, Drumgoole R, Helma CH, Ticknor LO, Okinaka RT, Jackson PJ (2006) Characterization of Bacillus cereus isolates associated with fatal pneumonias: strains are closely related to Bacillus anthracis and harbor B. anthracis virulence genes. J Clin Microbiol 44:3352–3360

    Article  CAS  Google Scholar 

  • Hu X, Swiecicka I, Timmery S, Mahillon J (2009) Sympatric soil communities of Bacillus cereus and Bacillus thuringiensis: population structure and potential plasmid dynamics of pXO1- and pXO2-like elements. FEMS Microbiol Ecol 70:344–355

    Article  CAS  Google Scholar 

  • Jernigan DB, Raghunathan PL, Bell BP, Brechner R, Bresnitz EA, Butler JC, Cetron M, Cohen M, Doyle T, Fischer M, Greene C, Griffith KS, Guarner J, Hadler JL, Hayslett JA, Meyer R, Petersen LR, Phillips M, Pinner R, Popovic T, Quinn CP, Reefhuis J, Reissman D, Rosenstein N, Schuchat A, Shieh WJ, Siegal L, Swerdlow DL, Tenover FC, Traeger M, Ward JW, Weisfuse I, Wiersma S, Yeskey K, Zaki S, Ashford DA, Perkins BA, Ostroff S, Hughes J, Fleming D, Koplan JP, Gerberding JL, National Anthrax Epidemiologic Investigation Team (2002) Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Infect Dis 8:1019–1028

    Google Scholar 

  • Kim K, Seo J, Wheeler K, Park C, Kim D, Park S, Kim W, Chung SI, Leighton T (2005) Rapid genotypic detection of Bacillus anthracis and the Bacillus cereus group by multiplex real-time PCR melting curve analysis. FEMS Immunol Med Microbiol 43:301–310

    Article  CAS  Google Scholar 

  • Klee SR, Nattermann H, Becker S, Urban-Schriefer M, Franz T, Jacob D, Appel B (2006) Evaluation of different methods to discriminate Bacillus anthracis from other bacteria of the Bacillus cereus group. J Appl Microbiol 100:673–681

    Article  CAS  Google Scholar 

  • Kumar R, Singh SK, Koshkin AA, RAjwanshi VK, Meldgaard M, Wengel J (1998) The first analogues of LNA (locked nucleic acids): phosphorothioate-LNA and 2′thio-LNA. Bioorg Med Chem Lett 8:2219–2222

    Article  CAS  Google Scholar 

  • Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, Singer MJ, Walburger DK, Lokhov SG, Gall AA, Dempcy R, Reed MW, Meyer RB, Hedgpeth J (2000) 3′-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res 28:655–661

    Article  CAS  Google Scholar 

  • Leask A, Delpech V, McAnulty J (2003) Anthrax and other suspect powders: initial responses to an outbreak of hoaxes and scares. N S W Public Health Bull 14:218–221

    Article  Google Scholar 

  • Lecouvet F, Irenge L, Vandercam B, Nzeusseu A, Hamels S, Gala JL (2004) The etiologic diagnosis of infectious discitis is improved by amplification-based DNA analysis. Arthritis Rheum 50:2985–2994

    Article  CAS  Google Scholar 

  • Marston CK, Gee JE, Popovic T, Hoffmaster AR (2006) Molecular approaches to identify and differentiate Bacillus anthracis from phenotypically similar Bacillus species isolates. BMC Microbiol 6:22

    Article  Google Scholar 

  • Mehrotra S, Balaram H (2007) Methanocaldococcus jannaschii adenylosuccinate synthetase: studies on temperature dependence of catalytic activity and structural stability. Biochemistry 46:12821–12832

    Article  CAS  Google Scholar 

  • Mouritzen P, Nielsen AT, Pfundheller HM, Choleva Y, Kongsbak L, Moller S (2003) Single nucleotide polymorphism genotyping using locked nucleic acid (LNA). Expert Rev Mol Diagn 3:27–38

    Article  CAS  Google Scholar 

  • Olsen JS, Skogan G, Fykse EM, Rawlinson EL, Tomaso H, Granum PE, Blatny JM (2007) Genetic distribution of 295 Bacillus cereus group members based on adk-screening in combination with MLST (multilocus sequence typing) used for validating a primer targeting a chromosomal locus in B. anthracis. J Microbiol Methods 71:265–274

    Article  CAS  Google Scholar 

  • Pannucci J, Okinaka RT, Sabin R, Kuske CR (2002) Bacillus anthracis pXO1 plasmid sequence conservation among closely related bacterial species. J Bacteriol 184:134–141

    Article  CAS  Google Scholar 

  • Park SH, Kim HJ, Kim JH, Kim TW, Kim HY (2007) Simultaneous detection and identification of Bacillus cereus group bacteria using multiplex PCR. J Microbiol Biotechnol 17:1177–1182

    CAS  Google Scholar 

  • Patra G, Sylvestre P, Ramisse V, Thérasse J, Guesdon JL (1996) Isolation of a specific chromosomic DNA sequence of Bacillus anthracis and its possible use in diagnosis. FEMS Immunol Med Microbiol 15:223–231

    Article  CAS  Google Scholar 

  • 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 67:3720–3727

    Article  CAS  Google Scholar 

  • 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 145:9–16

    Article  CAS  Google Scholar 

  • 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 47:693–699

    CAS  Google Scholar 

  • Skottman T, Piiparinen H, Hyytiäinen H, Myllys V, Skurnik M, Nikkari S (2007) Simultaneous real-time PCR detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis. Eur J Clin Microbiol Infect Dis 26:207–211

    Article  CAS  Google Scholar 

  • Thompson JD, Higgins DG, Gibson TJ, Clustal W (1994) Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

    Article  CAS  Google Scholar 

  • Valjevac S, Hilaire V, Lisanti O, Ramisse F, Hernandez E, Cavallo JD, Pourcel C, Vergnaud G (2005) Comparison of minisatellite polymorphisms in the Bacillus cereus complex: a simple assay for large-scale screening and identification of strains most closely related to Bacillus anthracis. Appl Environ Microbiol 71:6613–6623

    Article  CAS  Google Scholar 

  • Van Ert MN, Easterday WR, Simonson TS, U’Ren JM, Pearson T, Kenefic LJ, Busch JD, Huynh LY, Dukerich M, Trim CB, Beaudry J, Welty-Bernard A, Read T, Fraser CM, Ravel J, Keim P (2007) Strain-specific single-nucleotide polymorphism assays for the Bacillus anthracis Ames strain. J Clin Microbiol 45:47–53

    Article  Google Scholar 

  • Wong ML, Medrano JF (2005) Real-time PCR for mRNA quantification. Biotechniques 39:1–11

    Article  Google Scholar 

  • Zilinskas RA (1997) Iraq’s biological warfare program: the past as future? JAMA 278:418–424

    Article  CAS  Google Scholar 

Download references

Acknowledgments

This project was funded by the Department Management of Scientific and Technological Research of Defence (IRSD-RSTD, Royal High Institute for Defence) supporting research and development (grants MED-03 and MED-08), by the Walloon Region (grant 616313). We thank Yann Deccache (IRSD-RSTD), Elodie Carlier (IRSD-RSTD), Cathy Delcorps (IRSD-RSTD), and Michèle Bouyer (IRSD-RSTD) for their outstanding contribution to this work. We also thank Dr Angelo Madonna (Dugway Army Proving Ground, Utah, USA) for preparation of environmental samples containing killed Bacillus anthracis spores. Contributions of partner (3), (4), (6), and (7) to this work have been provided within the framework of the project EMS 4/EDA Database for Biological Agents. Contribution of partner (5) to this work has been provided in the framework of COSTB28.

Conflict of interest

None

Author information

Authors and Affiliations

  1. Defence Laboratories Department, Belgian Armed Forces, Brussels, Belgium

    Léonid M. Irenge & Jean-Luc Gala

  2. Centre for Applied Molecular Technologies, Université catholique de Louvain, Clos Chapelle-aux-Champs, 30 1200, Bruxelles, Belgium

    Léonid M. Irenge, Jean-François Durant & Jean-Luc Gala

  3. Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, 80937, Munich, Germany

    Herbert Tomaso

  4. Institute for Bacterial Infections and Zoonoses, Friedrich Loeffler Institut, Naumburgerstrasse 96a, 07743, Jena, Germany

    Herbert Tomaso

  5. Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, Postfach, CH-3001, Bern, Switzerland

    Paola Pilo

  6. Norwegian Defence Research Establishment (FFI), P.O. Box 25, 2027, Kjeller, Norway

    Jaran S. Olsen

  7. Centre d’Etudes du Bouchet (CEB), BP3, 91710, Vert le Petit, France

    Vincent Ramisse

  8. Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, Croix du Sud, 2/12, 1348, Louvain-la-Neuve, Belgium

    Jacques Mahillon

Authors
  1. Léonid M. Irenge
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jean-François Durant
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Herbert Tomaso
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Paola Pilo
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jaran S. Olsen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Vincent Ramisse
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jacques Mahillon
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jean-Luc Gala
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jean-Luc Gala.

Additional information

Léonid M. Irenge and Jean-François Durant contributed equally to this work.

Electronic supplementary material

Below is the link to the electronic supplementary material.

ESM Table 1

Microorganisms other than Bacillus used in this study (DOC 242 kb)

Fig. S4

Figures showing the real-time amplification curves obtained with the references DNA and with environmental samples from the SIBCRA exercises. a and b Amplification curves with LNA-BA1 probe (a) and LNA-BC2 probe (b) with serial dilutions of B. anthracis CEB 9434 DNA (from 500 pg to 50 fg). c Six amplification curves with LNA-BA1 probe of 13 of DNA extract from 13 environmental samples from the SIBCRA; d 13 amplification curves with LNA-BC2 probe of 13 DNA extracts from the same environmental samples. These amplification curves show the presence of six B. anthracis and of seven B. cereus group strains (non-B. anthracis) in these samples (DOC 3,239 kb)

Fig. S5

Alignment of purA gene sequences from 70 bacterial strains (DOC 411 kb)

Fig. S6

Alignment of ptsI gene sequences from 79 bacterial strains (DOC 396 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Irenge, L.M., Durant, JF., Tomaso, H. et al. Development and validation of a real-time quantitative PCR assay for rapid identification of Bacillus anthracis in environmental samples. Appl Microbiol Biotechnol 88, 1179–1192 (2010). https://doi.org/10.1007/s00253-010-2848-0

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-2848-0

Keywords

Search

Navigation