, 2008; López et al., 2010). At present, T. soleae is detected from fish by cultivation and subsequent identification using biochemical and serological techniques, which are frequently inconclusive and time-consuming. Moreover,

isolation from diseased fish is problematic because of the slow growth of the pathogen and the overgrowth and/or inhibition by other bacteria present within the lesions. PCR has proved to be useful for identification and detection of bacterial pathogens from samples without any need of cultivation (Cepeda et al., 2003; Gonzalez et al., 2003). The gene for the 16S rRNA is widely used in bacterial taxonomy as it contains variable stretches that have been used successfully for specific PCR primer design (Wiklund et al., 2000; Del Cerro et al., 2002; Oakey et al., 2003). However, it has been widely shown that the internal spacer region selleckchem (ISR) between Selumetinib in vivo the 16S and 23S rRNA genes is more variable between bacterial species than ribosomal genes themselves in both sequence and length (Barry et al., 1991; Hassan et al., 2003; Osorio et al., 2005). Species-specific primers derived from these sequences have also been reported (Kong et al., 1999; Lee et al., 2002; Hassan et al., 2008). In this study, we sequenced the ISR from T. soleae and designed species-specific primers, targeting both the 16S rRNA gene and ISR region, for its identification and detection

by PCR. The strains used in this study are listed in Table 1. Together with 32 reference strains, 57 isolates obtained in our laboratory from diseased flatfish were also used. These isolates were identified based on 16S gene sequencing and biochemical tests. All strains were cultured aerobically at 20 °C on tryptic soya agar (TSA) made with seawater, with the exception of those belonging to Tenacibaculum maritimum, which were grown on Flexibacter medium (FMM; Pazos et al., 1996).

Template DNA from pure cultures was prepared by boiling bacterial colonies for Buspirone HCl 10 min in distilled water followed by centrifugation at 12 400 g for 1 min to sediment the cell debris. DNA from tissue samples was extracted as follows: after homogenizing 100 mg of fish tissue in TE buffer (Sigma), SDS (1%) and proteinase K (100 μg mL−1) were added and the solution was incubated for 3 h or overnight at 56 °C. Thereafter, pancreatic RNAse (20 μg mL−1) was added and incubation was performed for 1 h at 37 °C. The solution was transferred to a phase-lock gel (Eppendorf) and the DNA was purified using the common phenol/chloroform/isoamyl alcohol procedure and finally precipitated with ethanol and dissolved in distilled water. The concentration and purity of genomic DNA were calculated from measurements of absorbance at 260 and 280 nm, recorded using a NanoDrop 1000 spectrophotometer. Partial 16S rRNA gene sequences were obtained using primers 20F and 1500R (Weisburg et al.