All these software perform smart designs where the probes have to be selected on the whole genomic DNA; this is the typical pipeline in contexts where no preselection of the target sequences has been made, which is not the case of ORMA. In fact, in both the presented datasets, the molecular complexity of the genomic material has been reduced by PCR on the 16S rRNA. The probe pairs design, then, was performed only on the basis of a specific subset of the whole 16S dataset, limited for the specific environment in which the target species have to be detected: cyanobacteria DNA was selected and amplified by cyano-specific PCR primers, while milk pathogens 16S rRNA sequences, although amplified by universal primers, were compared only to context-specific species. The double check in RDP and BLAST, performed after the complete probe pair design by ORMA, confirmed that our choice to work with such a reduced dataset was indeed correct, because the detected species accounted for the majority of the biological diversity present in the target matrix (i.e. milk). Moreover, many of the aforementioned software perform the specificity checks by extensive BLAST searches, which is a reasonable choice for designing specific probes starting from the whole genomic DNA; in case of datasets with limited complexity (or in which the complexity has been reduced by means of molecular procedures), this approach results too computationally intensive and unnecessary for the scope. ARB (21) and PRIMROSE (22) are tools widely used for the classification and the phylogenesis of bacterial species, structured for interacting with databases specific for the same molecular target (i.e. 16S rRNA) and operate a probe design on the basis of the phylogenesis of the species under analysis. None of these two software, however, is built specifically for the determination of discriminating positions within a set of very similar sequences and they provide probe design functionality only for hybridization assays or PCR primers. When used for probe design in detection application, the strategies are based on internal mismatches or on unique stretches of nucleotides (40) .