Intron splice site PCR analysis as a tool to discriminate Dekkera bruxellensis strains

Dekkera bruxellensis yeast species can develop off-flavours in wine through a specific reductive metabolism. In particular, volatile phenols are often produced in amounts that are higher than the perception threshold with a loss in product quality. Recent observations suggest that "brett spoilage" is strictly strain-dependent, and therefore, a rapid and reliable identification at strain level of D. bruxellensis becomes strategic for an efficient prevention. Among the techniques used to analyse DNA regions with high rate of sequence evolution, intron splice site PCR amplification (ISS-PCR) has allowed the detection of polymorphisms in commercial strains of S. cerevisiae. Recently, the genome of a D. bruxellensis isolated from wine has been sequenced and the results have shown that about 2% of the genes, a value similar to the ones found in other hemiascomycetes (1% in D. hansenii, 4% in S. cerevisiae) contain introns. Moreover, D. bruxellensis introns have 5′, 3′ and branch motifs that are very similar to the consensus motif in S. cerevisiae. Although the use of the 5′ intron-exon splice site as a target for ISS-PCR in D. bruxellensis did not allow the discrimination at strain level, an optimisation of primers could permit the development of a consistent tool for the typing of the species. In the present study, 17 D. bruxellensis strains belonging to the international CBS collection have been investigated for the ISSs, employing specific oligonucleotides containing different 5' consensus sequences: GTATGT (S. cerevisiae) and GTAAGT (D. bruxellensis). Results have shown that almost the whole yeast collection was discriminated at strain level using different combinations of primers. Therefore, to simplify the approach, a multiplex PCR protocol able to generate stable genetic profiles was developed