pestis specific virulence plasmids pPCP1 and pMT1. The plasmid pCD1 was not used as it is shared by other pathogenic Yersinia species. A chromosomal sequence of unknown function that had been identified using comparative genome hybridization [17] was selected as Y. pestis specific chromosomal target. SB203580 molecular weight Spores of B. thuringiensis were used as internal

control, not only for DNA amplification but also for successful DNA extraction. This member of the B. cereus group is closely related to B. anthracis and forms similar spores, while it contains species-specific plasmids. The B. thuringiensis plasmid gene encoding insecticidal crystal proteins (cry genes) was used as the signature sequence for the detection of DNA released from this organism’s spores. Sequence analysis tools, bioinformatics software Sequences retrieved from NCBI/EMBL were organized and aligned using the software package Kodon (Applied Maths, Ghent, Belgium). Comprehensive sequence alignments were made by performing BLAST searches from the selected targets to make sure all available sequence homologues were included in the alignments. Oligonucleotides for multiplex qPCR assays and for conventional PCR assays were designed using the software package Visual Oligonucleotide Modeling Platform version 6 (DNA software Inc. Ann Arbor, USA). The design strategy for multiplex qPCR assays was as follows. First, a hydrolysis

probe and primer Selleck Akt inhibitor set were designed for the B. thuringiensis internal control. Then, for each selected signature sequence a hydrolysis probe was designed, followed by the design of the corresponding primer set. A different strategy was chosen for the B. anthracis assay, because its chromosomal target sspE has homologues in other Bacillus, notably the internal control B. thuringiensis. To make sure that detection of B. anthracis sspE was highly selective, the exact positions of probe and primers were guided based on visual inspection of the alignment. Probe and primers were located in regions with mismatches Endonuclease between Bacillus species (notably between B. thuringiensis and B. anthracis),

and the primers were designed such that mismatch positions were located at their highly discriminating 3′-ends. Oligonucleotides that were calculated by the design software were first checked against the consensus alignment to exclude designs not covering all sequence variants, and were then evaluated using the simulation module of Visual OMP. All oligonucleotides designed were validated in silico by using BLAST searches in general and microbial genomes databases (NCBI/EMBL). Sequencing Sequences were obtained from the cry1 gene from B. thuringiensis strain ATCC 29730 and from the sspE gene from all B. anthracis strains in our culture collection, B. thuringiensis ATCC 29730 and B. cereus strains WSBC 10583, 10619, 10766, 10483, 10572, 10705, 10770 and 10865 (Additional file 1 Table S1).