ORMA: a tool for identification of species-specific variations in 16S rRNA gene and oligonucleotides design

Severgnini, Marco; Cremonesi, Paola; Consolandi, Clarissa; Caredda, Giada; De Bellis, Gianluca; Castiglioni, Bianca

Milk-pathogen dataset experiment. Milk pathogensrelated 16S sequences were retrieved from RDP-Ribosomal Database Project II (release 9.51, http:// rdp.cme.msu.edu/) (27) for a total of 738 sequences and divided into 13 subgroups, according to their phylogenetic classification. Only sequences of length >1200 bp and flagged as of 'good' quality were retrieved. Each subgroup was aligned independently in ClustalW, since the overall number of 16S sequences was >500 (above the maximum limit of the alignment tool) and imported into ORMA. The consensus sequence for each group was calculated with the same parameters specified for the cyanobacteria data set. Then, a new multiple-alignment step was performed before proceeding to actual probe design. One probe pair for each of the main six subspecies of the Streptococcus group (Streptococcus agalactiae, S. bovis, S. equi, S. canis, S. dysgalactiae S. uberis) was designed; moreover, the Staphylococcus aureus probe pair was designed independently from all the remaining coagulase negative Staphylococci (grouped in the 'Staphylococcus, no aureus' probe), because of its relationship with outbreaks of mastitis in dairy ruminants (28) and with major health issues, like food-related intoxications (29) . In order to have the best homogeneity among the species within each group, the design was actually performed in three rounds: (a) Salmonella spp. was aligned against Escherichia coli and related spp. consensus sequence only; (b) S. canis was aligned against Streptococcus group sequences only; (c) All the remaining positions were selected considering the alignment of all other subspecies. One probe pair per species was designed, except for Campylobacter spp. for which two probe pairs were evaluated in terms of reproducibility and specificity. The thermodynamic parameters were the same described for the cyanobacteria data set, except for the melting temperature, which was required to be in the range 67-698C. The inter-group score of the candidates was required to be above a threshold of 80%, as in the cyanobacteria dataset. Probe pair specificity was checked by both RDP II database and BLAST (Basic Local Alignment Search Tool, http://www.ncbi.nlm.nih.gov/ blast/Blast.cgi) (30) analysis, carefully examining the 3 0 -region of the discriminating probe, in order to exclude any interaction between probe pairs targeting different species. LDR probe pairs were mixed at a final concentration of 1 pmol/ml and tested on 13 DNAs from ATCC reference strains (LGC Promochem, Middlesex, UK) and bacterial collections (Supplementary Table 1 ). Genomic DNA was extracted following the protocol described by (31) , PCR amplified and analyzed in duplicate, by separated LDR reactions.