Padlock probes targeting the ITS region were designed and were ordered from Invitrogen Inc. (Breda, the selleck inhibitor Netherlands).

To optimise the binding efficiency to target DNAs, the padlock probes were designed with minimum secondary structure and with Tm of the 5′ end probe binding arm close to or above ligation temperature (63 °C, see below). To increase its discriminative specificity, the 3′ end binding arm was designed with a Tm 10–15 °C below ligation temperature. Linker regions of species-specific probes were taken from Zhou et al. [20] and 5′ and 3′ binding arms were designed in this article (Table 2). Oligonucleotide probes (Table 2) consisted of two adjacent complementary target sequences (12–26 bp) with a spacer region (63 bp) to facilitate loop formation and to provide a template for RCA primer binding. All primers and probes were synthesised by a commercial manufacturer (Invitrogen, Carlsbad,

CA, USA). One microlitre of ITS amplicon was mixed with 0.1 μl of ampligase (5 U μl−1), 2 nmol of padlock probe, 1 μl of 10× ligation buffer, 4.9 μl of water with a total reaction volume of 10 μl. Padlock probe ligation was conducted with one cycle of denaturation for 5 min at 95 °C, followed by seven cycles of 95 °C for 30 s and 4 min ligation at 63 °C. Exonucleolysis is required to remove unligated padlock probe and template PCR product and thus reduce subsequent ligation-independent amplification events. This step seems optional in previous works,[17] and we decided to delete this step without jeopardising Trichostatin A purchase speed and reliability of the method. Three microlitre of ligation product was used

as template for RCA. The total volume was 46 μl containing 1 μl Bst DNA polymerase LF (New England Biolabs, Hitchin, UK), 1 μl deoxynucleoside triphosphate mix (5 mmol l−1), 1.5 μl of 10 pmol of RCA primer each, 5 μl RCA buffer 10×, 36 μl water. Probe signals were amplified by incubation at 65 °C for 60 min, and accumulation of double stranded DNA products was visualised on a 1% agarose gel to verify the specificity of probe-template binding. Positive reactions showed a ladder-like pattern, whereas negative reactions showed a clean background. Smart DNA ladder (0.2–10 kb; Eurogentec, Seraing, Belgium) was used as molecular weight standard. To evaluate the detection limit of the RCA assay, two microlitres of each 10-fold serial dilution Sitaxentan was used in each RCA reaction. ITS amplicons of R. arrhizus var. delemar CBS 395.54 was used (Fig. 2). The ITS alignment revealed suitable positions for the development of padlock probes distinguishing between six taxa tested in this study. All tested strains generated positive results with respective padlock probes. The duration of the RCA assay was 2 h. Positive responses proved to be 100% specific for all strains, species-specific probes correctly identifying all six species and varieties analysed. No cross reaction was observed between these taxa (Fig. 1). CBS 395.54, CBS 109939, CBS 109940, CBS 103.