Applied Microbiology and Biotechnology

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

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: jean-luc. [email protected]

These authors contributed equally to this work. Online Resource Figure 5: alignment of purA gene sequences from 70 bacterial strains

Primers and LNA-probe positions in the purA gene alignment: blue color = forward primer (PUR-ANT-FOR); yellow color = fluorogenic LNA-probe (LNA-BA1) and pink color = reverse primer (PUR-ANT-REV).

EFLA = Enterococcus flavescens strain DSMZ7370; ECAS = Enterococcus casseliflavus strain DSMZ 20680; EVIL = Enterococcus villorum strain CODA-CERVA; EDUR = Enterococcus durans strain DSMZ20633; EFCL = Enterococcus faecalis strain

DSMZ2570; EGAL = Enterococcus gallinarum strain DSMZ20628; ERAF = Enterococcus raffinosus strain DSMZ75633; EAVI =

Enterococcus avium strain DSMZ20679; 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; SSUI = Streptococcus suis strain DSMZ9682; SGOR = Streptococcus gordonii strain

DSMZ6777; EHIR = Enterococcus hirae strain DSMZ20160; EFCM = Enterococcus faecium strain DSMZ6177; SMUT =

Streptococcus mutans strain DSMZ20523; SPNE = Streptococcus pneumoniae strain DSMZ20566; SMIT = Streptococcus mitis strain DSMZ12643; SORA = Streptococcus oralis strain DSMZ20627; STHE = Streptococcus thermophilus strain DSMZ20617;

LLAC = Lactococcus lactis strain DSMZ20481; SEPI = Staphylococcus epidermidis strain ATCC14990; SCAPCAP =

Staphylococcus capitis capitis strain ATCC27840; SHOM = Staphylococcus hominis strain ATCC27844; SHAE = Staphylococcus haemolyticus strain ATCC29970; SLUG = Staphylococcus lugdunensis strain ATCC43809; SAUR = Staphylococcus aureus strain

ATCC35884; SWAR = Staphylococcus warneri strain ATCC27836 ; SCAPURE = Staphylococcus capitis ureolyticus strain ATCC49326; SXYL = Staphylococcus xylosus strain ATCC35663; SSAP = Staphylococcus saprophyticus strain ATCC15305; SGAL

= Staphylococcus gallinarum strain ATCCC3572; SSCI = Staphylococcus sciuri strain ATCC29062; SSIM = Staphylococcus simulans strain ATCC27848; SCOHCOH = Staphylococcus cohnii cohnii strain ATCC35662; SCAS = Staphylococcus caseolyticus strain ATCC13548; SSCH = Staphylococcus schleiferi schleiferi strain ATCC43808; SAURICU = Staphylococcus auricularis strain

ATCC33753; BSPH = Bacillus sphaericus strain NCTC2608; BSUB = Bacillus subtilis strain DSMZ1092; BLIC = Bacillus licheniformis strain DSMZ2048; BPUM = Bacillus pumilus strain WSBC10031; BMEG = Bacillus megatherium strain WSBC10380;

BCER = Bacillus cereus strain ATCC10987; BTHUISR = Bacillus thuringiensis serovar Israelensis strain UCL 4Q2-72;

BCMYC003 = Bacillus mycoides strain UCL MYC003; BCMYC004 = Bacillus mycoides strain UCL MYC004; BCMYC005 =

Bacillus mycoides strain UCL MYC005; BCNRS306-2 = Bacillus mycoides strain UCL NRS306; BKKK = Bacillus thuringiensis serovar Konkunkian strain CEB 97-27; BANT9434 = Bacillus anthracis strain CEB 9434; BCNRS1216 = Bacillus mycoides strain

UCL NRS1216; BWEI = Bacillus mycoides strain UCL WSBC10240; BCKNC1-2 = Bacillus mycoides strain UCL KNC1-2;

BCBD23 = Bacillus mycoides strain UCL BD23; BCKBS1-14 = Bacillus mycoides strain UCL KBS1-14; BCKBS2-12 = Bacillus mycoides strain UCL KBS2-12; BCKBS317 = Bacillus mycoides strain UCL KBS317; BCB3436 = Bacillus mycoides strain UCL

B3436; BCBD5 = Bacillus pseudomycoides strain UCL BD5; BCNRS321 = Bacillus mycoides strain UCL NRS321; BCKNC2-18 =

Bacillus mycoides strain UCL KNC2-18; BCKNC2-16 = Bacillus mycoides strain UCL KNC2-16; BCKNC1-10 = Bacillus mycoides strain UCL KNC1-10; BTHUKUR = Bacillus thuringiensis serovar Kurstaki strain UCL T03A016; BCKNC2-13 = Bacillus mycoides strain UCL KNC2-13; LMON = Listeria monocytogenes strain DSMZ20600 and LINN = Listeria innocua strain

DSMZ20649.