<p>Development and validation of a Real-time quantitative PCR assay for Rapid Identification of Bacillus anthracis in environmental samples</p><p>Applied Microbiology and Biotechnology</p><p>Léonid M. Irenge1,2 , Jean-François Durant2 , Herbert Tomaso3,4, Paola Pilo5, Jaran S. Olsen6, Vincent Ramisse7, Jacques</p><p>Mahillon8 and Jean-Luc Gala1,2*</p><p>1. Defence Laboratories Department, Belgian Armed Forces, Brussels, Belgium.</p><p>2. Centre for Applied Molecular Technologies, Université catholique de Louvain, Clos Chapelle-aux-Champs, 30 B-1200 Bruxelles,</p><p>Belgium</p><p>3. Bundeswehr Institute of Microbiology, Neuherbergstrasse 11, 80937 Munich, Germany</p><p>4. Institute for Bacterial Infections and Zoonoses, Friedrich Loeffler Institut, Naumburgerstrasse 96a, 07743 Jena, Germany</p><p>5. Institute of Veterinary Bacteriology, Vetsuisse Faculty, University of Bern, Länggassstrasse 122, Postfach, CH-3001 Bern,</p><p>Switzerland</p><p>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</p><p>8. Laboratory of Food and Environmental Microbiology, Université catholique de Louvain, Croix du Sud, 2/12, B-1348 Louvain-la-</p><p>Neuve, Belgium</p><p>*Corresponding author: Jean-Luc Gala, Centre for Applied Molecular Technologies, UCL/30.46, Clos Chapelle-aux-Champs, 30 B-</p><p>1200 Bruxelles, Belgium</p><p>Tel: +32 2 764 31 65, Fax: +32 2 764 31 66 - E-mail: jean-luc. [email protected] </p><p>These authors contributed equally to this work. Online Resource Figure 5: alignment of purA gene sequences from 70 bacterial strains</p><p>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). </p><p>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 </p><p>DSMZ2570; EGAL = Enterococcus gallinarum strain DSMZ20628; ERAF = Enterococcus raffinosus strain DSMZ75633; EAVI = </p><p>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 =</p><p>Streptococcus bovis strain DSMZ20480; SSUI = Streptococcus suis strain DSMZ9682; SGOR = Streptococcus gordonii strain </p><p>DSMZ6777; EHIR = Enterococcus hirae strain DSMZ20160; EFCM = Enterococcus faecium strain DSMZ6177; SMUT = </p><p>Streptococcus mutans strain DSMZ20523; SPNE = Streptococcus pneumoniae strain DSMZ20566; SMIT = Streptococcus mitis strain DSMZ12643; SORA = Streptococcus oralis strain DSMZ20627; STHE = Streptococcus thermophilus strain DSMZ20617; </p><p>LLAC = Lactococcus lactis strain DSMZ20481; SEPI = Staphylococcus epidermidis strain ATCC14990; SCAPCAP = </p><p>Staphylococcus capitis capitis strain ATCC27840; SHOM = Staphylococcus hominis strain ATCC27844; SHAE = Staphylococcus haemolyticus strain ATCC29970; SLUG = Staphylococcus lugdunensis strain ATCC43809; SAUR = Staphylococcus aureus strain </p><p>ATCC35884; SWAR = Staphylococcus warneri strain ATCC27836 ; SCAPURE = Staphylococcus capitis ureolyticus strain ATCC49326; SXYL = Staphylococcus xylosus strain ATCC35663; SSAP = Staphylococcus saprophyticus strain ATCC15305; SGAL</p><p>= 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 </p><p>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;</p><p>BCER = Bacillus cereus strain ATCC10987; BTHUISR = Bacillus thuringiensis serovar Israelensis strain UCL 4Q2-72; </p><p>BCMYC003 = Bacillus mycoides strain UCL MYC003; BCMYC004 = Bacillus mycoides strain UCL MYC004; BCMYC005 = </p><p>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 </p><p>UCL NRS1216; BWEI = Bacillus mycoides strain UCL WSBC10240; BCKNC1-2 = Bacillus mycoides strain UCL KNC1-2; </p><p>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 </p><p>B3436; BCBD5 = Bacillus pseudomycoides strain UCL BD5; BCNRS321 = Bacillus mycoides strain UCL NRS321; BCKNC2-18 = </p><p>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 </p><p>DSMZ20649. </p>