Development and validation of a Real-time quantitative PCR assay for Rapid Identification of Bacillus anthracis in environmental samples

Applied Microbiology and Biotechnology

Léonid M. Irenge1,2 , Jean-François Durant2, Herbert Tomaso3,4, Paola Pilo5, Jaran S. Olsen6, Vincent Ramisse7, Jacques Mahillon8 and Jean-Luc Gala1,2*

  1. Defence Laboratories Department, Belgian Armed Forces, Brussels, Belgium.
  2. Centre for Applied Molecular Technologies, Université catholique de Louvain, Clos Chapelle-aux-Champs, 30 B-1200 Bruxelles, Belgium
  3. Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, 80937 Munich, Germany
  4. Institute for Bacterial Infections and Zoonoses, Friedrich Loeffler Institut, Naumburgerstrasse 96a, 07743 Jena, Germany
  5. Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, Postfach, CH-3001 Bern, Switzerland
  6. Norwegian Defence Research Establishment (FFI) P.O. Box 25, N-2027 Kjeller, Norway
  7. Centre d’Etudes du Bouchet (CEB), BP3, 91710 Vert le Petit, France
  8. Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, Croix du Sud, 2/12, B-1348 Louvain-la-Neuve, Belgium

*Corresponding author: Jean-Luc Gala, Centre for Applied Molecular Technologies, UCL/30.46, Clos Chapelle-aux-Champs, 30 B-1200 Bruxelles, Belgium

Tel: +32 2 764 31 65, Fax: +32 2 764 31 66 - E-mail:

These authors contributed equally to this work.

Online Resource Figure 6: alignment of ptsI gene sequences from 79 bacterial strains

Primers and LNA-probe positions in the ptsI gene alignment: blue color = forward primer (PTS-BAC-FOR); yellow color = fluorogenic LNA-probe (LNA-BC2) and pink color = reverse primer (PTS-BAC-REV).

BCBD23 = Bacillus mycoides strain UCL BD23; BCBD3 = Bacillus mycoides strain UCL BD3; BCKBS2-12 = Bacillus mycoides strain UCL KBS2-12;BCKNC3-1 = Bacillusmycoides strain UCL KNC3-1;BTHUISR = Bacillus thuringiensis serovar Israelensis strain UCL 4Q2-72;BCER10987 = Bacillus cereus strain ATCC10987;BANT = Bacillus anthracis strain CEB 9434;BKKK = Bacillus thuringiensisserovar Konkunkian strain CEB 97-27;BWEI = Bacillus mycoides strain UCL WSBC10240;BCBD2 = Bacillus mycoides strain UCL BD2;BCKNC1-2 = Bacillus mycoides strain UCL KNC1-2;BCKBS1-14 = Bacillus mycoides strain UCL KBS1-14; BCKBS317 = Bacillus mycoides strain UCL KBS317;BCNRS306 = Bacillus mycoides strain UCL NRS306;BCNRS1216 = Bacillus mycoides strain UCL NRS1216;BCMYC003 = Bacillus mycoides strain UCL MYC003; BCMYC004 = Bacillus mycoides strain UCL MYC004; BCMYC005 = Bacillus mycoides strain UCL MYC005;BTHUKUR = Bacillus thuringiensisSerovar Kurstaki strain UCL T03A016;BCNRS323 = Bacillus mycoides strain UCL NRS323;BCKNC1-10 = Bacillus mycoides strain UCL KNC1-10;BCKNC2-16 = Bacillus mycoides strain UCL KNC2-16;BCKNC2-18 = Bacillus mycoides strain UCL KNC2-18; BCNRS321 = Bacillus mycoides strain UCL NRS321;BCKNC1-16 = Bacillus mycoides strain UCL KNC1-16;BCKNC2-13 = Bacillus mycoides strain UCL KNC2-13;BCBD5 = Bacilluspseudomycoides strain UCL BD5;BPOL = Bacillus polymyxa strain DSMZ2048; BSPH = Bacillus sphaericus strain NCTC2608;BSTE = Bacillus stearothermophilusstrain B287; BSUB = Bacillus subtilis strain DSMZ1092;BLIC = Bacillus licheniformis strain DSMZ2048; BPUM = Bacillus pumilus strain WSBC10031;BFIR = Bacillus firmus strain DSMZ2899; BMEG = Bacillus megatherium strain WSBC10380;BLAT = Bacillus laterosporum ATCC9141; BALV = Bacillus alvei DSMZ50905; BCIR = Bacillus circulans DSMZ8579; BCOA = Bacillus coagulans DSMZ1103; EFLA = Enterococcus flavescens strain DSMZ7370; ECAS = Enterococcus casseliflavus strain DSMZ 20680;EFCL = Enterococcus faecalis strain DSMZ2570;EVIL = Enterococcus villorum strain CODA-CERVA; EDUR = Enterococcus durans strain DSMZ20633; EGAL = Enterococcus gallinarum strain DSMZ20628; ERAF = Enterococcus raffinosus strain DSMZ75633; EAVI = Enterococcus avium strain DSMZ20679; LLAC = Lactococcus lactis strain DSMZ20481; SAGA = Streptococcus agalactiae strain DSMZ2134; SSAN = Streptococcus sanguinis strain DSMZ20567, SPYO = Streptococcus pyogenes strain DSMZ20565; SCAN = Streptococcus canis strain DSMZ20386; SBOV = Streptococcus bovis strain DSMZ20480; SGOR = Streptococcus gordonii strain DSMZ6777; EHIR = Enterococcushirae strain DSMZ20160; EFCM = Enterococcus faecium strain DSMZ6177; SPNE = Streptococcus pneumoniae strain DSMZ20566; SMIT = Streptococcus mitis strain DSMZ12643; SORA = Streptococcus oralis strain DSMZ20627; SMUT = Streptococcus mutans strain DSMZ20523; SSUI = Streptococcus suis strain DSMZ9682; STHE = Streptococcus thermophilus strain DSMZ20617; SHOM = Staphylococcus hominisstrain ATCC27844; SHAE = Staphylococcus haemolyticusstrain ATCC29970; SLUG = Staphylococcus lugdunensisstrain ATCC43809; SEPI = Staphylococcus epidermidis strain ATCC14990; SCAPCAP = Staphylococcus capitiscapitisstrain ATCC27840;SWAR = Staphylococcus warneristrain ATCC27836;SAUR = Staphylococcus aureusstrain ATCC35884;SGAL = Staphylococcus gallinarum strain ATCCC3572;SCAPURE = Staphylococcus capitis ureolyticusstrain ATCC49326;SSAP = Staphylococcus saprophyticus strain ATCC15305;SSCH = Staphylococcus schleiferi schleiferistrain ATCC43808;SSCI = Staphylococcus sciuristrain ATCC29062;SCOHCOH = Staphylococcus cohnii cohniistrain ATCC35662;SCAS = Staphylococcus caseolyticusstrain ATCC13548;LMON = Listeria monocytogenes strain DSMZ20600,LINN = Listeria innocua strain DSMZ20649; ECOK12 = Escherichia coli strain K12 DSMZ6367.