DNA was then extracted from washed ectomycorrhizae by NucleoSpin Plant II DNA extraction kit (Macherey-Nagel GmbH & Co. KG) and from soil (250-mg sample) by NucleoSpin Soil DNA kit (Macherey-Nagel GmbH & Co. KG) as indicated above. The total DNA concentration in extracts is given in Appendix S1, sheet ‘Field detection’. Undiluted DNA extracts were amplified in nested PCR (first run with the NSI1/NLB4 primer pair, second run with the Tu1sekvF/Tu2sekvR
primer pair, annealing at 59 °C) and cleaved by TaiI restriction endonuclease as described above. Two sequence motifs, common for T. aestivum but not present in ITS region of other Tuber spp. and other identified organisms in GenBank, were found. Two primers targeting these motifs were then designed. According to the analysis of GenBank data, the virtual length of the PCR product amplified using this primer pair Selleck Vemurafenib is 496–502 bp. The primers binding to 17 bp motifs were called Tu1sekvF (forward, its target motif is localized in ITS1) and Tu2sekvR (reverse, target motif localized in ITS2) (for nucleotide sequence see Table 1). The motifs have 100% homology to corresponding sites in all studied GenBank ITS sequences
of T. aestivum and Tuber uncinatum with the exception of the sequence AJ492216, showing one gap in the motif recognized by the primer Tu1sekvF, and sequence AJ888120, possessing one substitution Gefitinib price in the motif recognized by the primer Tu2sekvR (Appendix S4). As seen in Table 2, primer pair tubtubf/elytubr designed to amplify Tuber spp. amplified DNA from almost all the samples, indicating good quality DNA extracts. Only two samples (Tuber bellonae and one sample of Tuber rufum) gave no signal. In general, all three primer pairs supposedly specific to T. aestivum showed
some nonspecific amplification of nontarget species DNA. Direct PCR with negative controls A–E showed that the primer pair UncI/UncII was prone to nonspecific DNA amplification. The same trend was noted in the case of primer pair tubtubf/elytubr working at an annealing temperature lower than that recommended by the designers (Zampieri et al., 2009) to increase Cediranib (AZD2171) its sensitivity to T. aestivum. The primer pair BTAE-F/BTAEMB-R seems to be the most robust to nonspecific amplification and the pair Tu1sekvF/Tu2sekvR is intermediate in this regard. Nested PCR with nontarget DNA samples always gave negative results (Table 2). In the test of the sensitivity to target DNA diluted in a large amount of nontarget DNA, nested PCR with primer pairs NSI1/NLB4 and Tu1sekvF/Tu2sekvR still gave a positive result if nontarget DNA contained 0.01% (1.25 pg per PCR reaction) of T. aestivum S13 DNA (see Appendix S5). Unfortunately, nested PCR using the primers BTAE-F and BTAEMB-R (Bt2a/BTAEMB-R in first amplification and BTAE-F/Bt2b in second amplification) was not successful. TaiI cleavage of T.