Italian industrial pasta has to be prepared using exclusively durum wheat (Triticum turgidum L. var. durum) semolina. The Italian law only allows the presence of maximum 3% soft wheat (Triticum aestivum L.) as an accidental contamination during the storage of raw material. Traditionally, the presence of soft wheat contamination is assessed through enzymatic methods, which are fairly cumbersome and do not allow an accurate quantification of the contaminant. Recently, a new generation of methods which employ DNA screening for sequences localized in the D-genome, characteristic for soft wheat, has become available, including real-time quantitative PCR analyses (Alorio et al. 2003; Terzi et al. 2003). In a previous study, a microsatellite sequence specific of the D genome had been chosen, among others, for traceability of soft wheat in qualitative analysis and quantitative Sybr green based real-time experiments (Pasqualone et al. 2007). However, a good quantification was only obtained for semolina samples as compared to processed food. In this work, we describe an improved method based on the same soft wheat genomic region, using an alternative approach. The DNA region was cloned and sequenced, and primers and a dual-labeled (Taqman(TM)) probe were designed nearby the microsatellite repeat. Semolina and pasta samples were prepared from several blends of durum wheat:soft wheat in controlled ratios, and genomic DNA extraction was optimized to recover a high DNA amount. The Taqman(TM) approach allowed to avoid aspecific amplifications due to primer dimer formation, and therefore produced a more accurate estimate of soft wheat contamination in semolina based products.
A Taqman based approach for the quantification of soft wheat adulterations in durum wheat pasta
Sonnante G;Morgese A;Pignone D;
2008
Abstract
Italian industrial pasta has to be prepared using exclusively durum wheat (Triticum turgidum L. var. durum) semolina. The Italian law only allows the presence of maximum 3% soft wheat (Triticum aestivum L.) as an accidental contamination during the storage of raw material. Traditionally, the presence of soft wheat contamination is assessed through enzymatic methods, which are fairly cumbersome and do not allow an accurate quantification of the contaminant. Recently, a new generation of methods which employ DNA screening for sequences localized in the D-genome, characteristic for soft wheat, has become available, including real-time quantitative PCR analyses (Alorio et al. 2003; Terzi et al. 2003). In a previous study, a microsatellite sequence specific of the D genome had been chosen, among others, for traceability of soft wheat in qualitative analysis and quantitative Sybr green based real-time experiments (Pasqualone et al. 2007). However, a good quantification was only obtained for semolina samples as compared to processed food. In this work, we describe an improved method based on the same soft wheat genomic region, using an alternative approach. The DNA region was cloned and sequenced, and primers and a dual-labeled (Taqman(TM)) probe were designed nearby the microsatellite repeat. Semolina and pasta samples were prepared from several blends of durum wheat:soft wheat in controlled ratios, and genomic DNA extraction was optimized to recover a high DNA amount. The Taqman(TM) approach allowed to avoid aspecific amplifications due to primer dimer formation, and therefore produced a more accurate estimate of soft wheat contamination in semolina based products.I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
