Cascade, CO, USA). Incompatibility among primers was avoided by in silico analysis of the formation of secondary structures, and oligonucleotides forming dimers with energy Selleck Dibutyryl-cAMP values lower than −6 kcal/mol and hairpins with Tm higher than 40C were discarded. The specificity of the oligonucleotides was first assessed by blastn (http://​www.​ncbi.​nlm.​nih.​gov/​blast/​Blast.​cgi?​PAGE=​Nucleotides). The reaction mix included 80 μg/tube of bovine serum albumin (Roche España, Madrid, Spain), 3.75 mM MgCl2 (Applied Biosystems), 200 μM dNTPs (Applied Biosystems) and 4U of AmpliTaq Gold® DNA Polymerase (Amersham Pharmacia Biotech, Cerdanyola del Vallès, Barcelona,

Spain). Primer concentrations ranged from 0.6 to 1 μM (Additional file 2: Table S2). The amplification cycles included an initial cycle of 94C for 9 min, followed by 40 cycles of 94C 30 s, 60C 1 min, and 72C 1 min, with a final extension at 72C for 10 min. The amplifications were performed in an MJ Research

Duvelisib PTC-200 (Bio-Rad Laboratories, S.A., Alcobendas, Madrid, Spain) in volumes of 50 μl. Hybridization by RLB was performed as described [25] using 48C for the hybridization and 40C for the conjugate and the washing steps. Concentration of probes ranged from 0.8 to 6.4 pmols/μl (Additional file 2: Table S2). Two overlapping films (SuperRX, Fujifilm España S.A., Barcelona, Spain), were used in each assay to obtain a less

and more exposed image for each membrane. Table 1 Scheme of the presence/absence of the Coxiella burnetii ORFs selected for the determination of genomic groups Target GGI GGII GGIII GGIV GGV GGVI GGVII GGVIII CBU0007 + + + − + + + + CBU 0071 + + + + − + + − CBU 0168 + + + − + + − + CBU 0598 + + − + + + + + CBU 0881 + + + + + − − − CBU 1805 + + + + − + + + CBU 2026 + − + + + + + + The sensitivity of the technique was checked with serial 10-fold dilutions of a purified DNA stock of the isolate Nine OSBPL9 Mile phase II (NMII) and the specificity was studied by subjecting to the method 104 genome equivalents of a selection of other bacterial species causing zoonoses or related illness (Orientia tsutsugamushi, Rickettsia conorii, R. typhi, Legionella pneumophila, Francisella tularensis subsp. holarctica, Bartonella henselae, Chlamydophila pneumoniae, and Mycoplasma pneumoniae). To assess the reproducibility of the methodology, DNA extracted from 2 different passages (n and n+10) of 5 reference isolates (NMI, CS-27, Priscilla, SQ217, F2) and a local isolate from cattle (273) (Additional file 1: Table S1) were analyzed. The results of the GT study were further analyzed by using InfoQuest™FP 4.50 (BioRad, Hercules, CA, USA). Clustering analyses used the binary coefficient (signaling pathway Jaccard) and UPGMA (Unweigthed Pair Group Method Using Arithmetic Averages) to infer the phylogenetic relationships.