Set up of a Real-Time PCR method for detecting and quantifying soft adulteration in durum wheat products. (Contributo in atti di convegno)

Type
Label
  • Set up of a Real-Time PCR method for detecting and quantifying soft adulteration in durum wheat products. (Contributo in atti di convegno) (literal)
Anno
  • 2008-01-01T00:00:00+01:00 (literal)
Alternative label
  • Pasqualone A., Montemurro C., Grinn-Gofron A., Sonnante G., Blanco A (2008)
    Set up of a Real-Time PCR method for detecting and quantifying soft adulteration in durum wheat products.
    in ICC International Conference – Bosphorus 2008, Istanbul-Turkey, 24-26 aprile 2008
    (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#autori
  • Pasqualone A., Montemurro C., Grinn-Gofron A., Sonnante G., Blanco A (literal)
Pagina inizio
  • 138 (literal)
Pagina fine
  • 138 (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#affiliazioni
  • Università di Bari, CNR-IGV (literal)
Titolo
  • Set up of a Real-Time PCR method for detecting and quantifying soft adulteration in durum wheat products. (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#isbn
  • 978-9944-0519-0-3 (literal)
Abstract
  • Italian law (1) prohibits the manufacture of pasta containing more than 3% soft wheat (Trilicum aestivum L) for the domestic market, while it allows import-export of pasta totally or partially prepared from soft wheat, in this case requiring to clearly indicate it in the label. Consequently, a strong interest towards the detection of soft wheat has stimulated the development of numerous analytical methods, generally aimed at searching specific protein fractions, the first set up as early as at the end of 1960s (2). More recently, a new generation of methods which employ DNA screening for sequences localized in the D-genome. characteristic for common wheat, has become available. Microsatellites are sequences of repeated DNA that can be analyzed by means of a single PCR reaction, providing short-sized arnplicons (3). The small size of generated amplicons (around 200 base pairs) is crucial if analyzing food samples submitted, during their production process, to high temperature and/ or strong mechanical treatments, such in case of pasta. The arm of this work was to evaluate the possibility to apply the analysis of DNA microsateUites to set up a method of detection of soft wheat in semolina and pasta. The first step consisted in DNA extraction from flour and semolina from various cultivars of soft and durum wheat (Triticum lurgidum L. var. durum), and from commercial pasta, by comparing CTAB laboratory protocol and the commercial kits NucleoSpin Plant (Macherey-Nagel), NucleoSpm Food (Macherey-Nagel) and Gene Elute Plant (Sigma). Their protocols have been modified to better adapt to the food sample considered. The better efficiency, in terms of extraction yield and DNA quality and purity, was obtained using NucleoSpin Food, which gave an average yield of 275 ug DNA/g sample. Then, nine primer pairs amplifying microsatellite sequences, chosen for being localized on D-genome according to literature data, were tested on the DNA extracted, with the aim of verifying their effectiveness in distinguishirsgsoft wheat from durum one. The obtained results enabled to select an efficient D-genome-specific microsatellite sequence able to detect common wheat. A polymorphism of the type \"presence/absence\" was observed, with no amplification in durum wheat (negative control), and an amplicon of 210 base pairs present solely in samples deriving from soft wheat. The first trials were carried out by qualitative PCR, able just to assess presence or absence of soft wheat with a threshold of 3%, but not adequately able to quantify it. Subsequently, semolina and pasta samples were prepared from blends of durum wheat:soft wheat in the ratios 60:40, 70:30, 80:20, 90:10, 95:5, 97:3, and 97.5:2.5, and SYBR Green real-time PCR was used to quantify the soft wheat adulteration. In this way the threshold was lowered to 2.5%. We are currently proceeding to a further improvement of the method by a TaqMan approach. (literal)
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