, 2002). PCR products were electrophoresed in a 1% agarose gel and purified with the kit GenElute PCR Clean-up (Sigma) following the manufacturer’s instructions. The purified products were cloned in pGEM-T
Easy Vector System II kit (Promega) or directly used for sequencing. Sequencing was accomplished using the kit BigDye Terminator v3.1 Cycle Sequencing (Applied Biosystems) and an ABI Prism 3130 DNA Sequencer (Applied Biosystems). The sequences were analyzed using chromaslite v2.01 and seqmanii (DNASTAR) programs and subjected to blast searches to retrieve the most closely related sequences. The presence of tRNA genes was determined using tRNAscan-SE 1.21 software (Lowe & Eddy, 1997). Previously reported 16S rRNA gene and ISR sequences from T. soleae and related species, retrieved from GenBank database (http://www.ncbi.nlm.nih.gov/genbank/) and those obtained in this study, were aligned by using the program clustalw (http://www.ebi.ac.uk/Tools/msa/clustalw2/) find more and examined for areas of similarity and variability between different species and strains. On the basis of the alignments, two variable regions were chosen and a pair of primers was designed by using the primer3 program Lumacaftor solubility dmso (http://frodo.wi.mit.edu/; Rozen & Skaletsky, 2000). Primers were synthesized by Thermo Scientific (Ulm, Germany). The PCR amplifications were carried out using the commercial
kit RedTaq ReadyMix (Sigma), which included all necessary reagents except the primers and DNA template. The PCR mixture consisted of reaction buffer (10 mmol L−1 Tris–HCl pH 8.3, 50 mmol L−1 KCl, 1.5 mmol L−1 MgCl2), 200 μmol L−1 Clomifene of each dNTP, 200 nmol L−1 of each primer,
3 U of Taq DNA polymerase, template DNA, and double-distilled water up to a final volume of 50 μL. The amplification was performed in a Mastercycler gradient (Eppendorf) as follows: an initial denaturation at 94 °C for 5 min followed by 45 amplification cycles (denaturation at 94 °C for 1 min, annealing at 57 °C for 45 s, and extension at 72 °C for 1 min), and a final elongation at 72 °C for 5 min. DNA from strain T. soleae a47 was included as a positive control and distilled water as a negative control. PCR products were electrophoresed on a 1% agarose TBE gel stained with SYBR Safe DNA Gel Stain (Invitrogen); a 1-kb DNA ladder (Biotools) was included as a molecular weight marker. To test the specificity of the primers in the PCR procedure, nine T. soleae strains, isolated from three different hosts and including the type strain, and 81 strains of other species, most of them taxonomically and/or ecologically related, were used as positive and negative controls, respectively (Table 1). For PCR amplification, 100 ng DNA template was used for each strain. The detection limit was evaluated using 10-fold serially diluted DNA, isolated from strain T. soleae a47, over the range 100 ng to 100 fg. Large amounts of DNA (0.5–3 μg) were also assayed.