16S rRNA sequences of pathogens contaminating bovine milk or related to bovine mastitis were used to design LDR oligonucleotide probes by ORMA, providing a further confirmation of its reliability and specificity. In this study, three round of design were actually performed, in order to have the best homogeneity between the species used in each round. A single round would have caused the loss of discriminating positions due to misalignment of some species (e.g. Salmonella) which are somehow different from all others. ORMA found a total of 392 candidate positions (34, 4 and 354 in the design for Salmonella, S. canis and all remaining species, respectively), which were selected according to the quality ranking scores assigned by ORMA. In this experiment, ORMA calculated only the intra-group score, but not the inter-group score, because of the fact that the sequences for each group were imported separately and the software was unable to recall the position corresponding to discriminating ones in all the sequences constituting each of the consensuses. The candidate probes were all characterized by an optimal specificity of the discriminating base, as suggested by the intra-group scores which were above 90% in 11 out of 14 cases. The scores were, in any case, above the fixed threshold of 80%, having an average of 94.0% AE 6.9% (n = 14). Also in this case, the lowest score (i.e. 80%) was that of the cluster (i.e. S. equi) constituted by the lowest number of sequences (n = 5). The final evaluation on the candidate probe pairs was made by RDP and BLAST checks, because of the multiplicity of species, whose 16S rRNA gene was amplified by means of universal primers, potentially present in milk-derived matrixes and the lack of a complete internal negative set in ORMA. The probes were slightly longer than the ones on cyanobacteria dataset, with an average length of about 40 nt, with very homogeneous melting temperatures (mean T m = 67.6 AE 0.4, n = 28) and a very low number of degenerated bases (only the DS probe for S. equi had 1 degenerate base) ( Table 3 ). The consensus scores for both the DS and CP confirmed the overall quality of the probes (average score of 96.5 AE 4.2, n = 28, with 60% of the probes having a score >99%).