DNA-aptamers for mycotoxins: application of ochratoxin A aptamer to wheat analysis. (Comunicazione a convegno)

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  • DNA-aptamers for mycotoxins: application of ochratoxin A aptamer to wheat analysis. (Comunicazione a convegno) (literal)
Anno
  • 2011-01-01T00:00:00+01:00 (literal)
Alternative label
  • De Girolamo A., Schena R., McKeague M., DeRosa M.C., Miller J.D., Visconti A. (2011)
    DNA-aptamers for mycotoxins: application of ochratoxin A aptamer to wheat analysis.
    in 5th International Symposium on “Recent Advances on Food Analysis”., Praga, Repubblica Ceca, 1-4 Novembre 2011
    (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#autori
  • De Girolamo A., Schena R., McKeague M., DeRosa M.C., Miller J.D., Visconti A. (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#note
  • Book of Abstracts: p. 122. (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#affiliazioni
  • ISPA-CNR Carleton University, Ottawa, CA (literal)
Titolo
  • DNA-aptamers for mycotoxins: application of ochratoxin A aptamer to wheat analysis. (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#isbn
  • 978-80-7080-795-8 (literal)
Abstract
  • Aptamers are single-stranded oligonucleotides that are mainly selected using SELEX (Systematic Evolution of Ligands by EXponential) enrichment and are able to discriminate target molecules with high affinity and specificity, even in the case of very closely related structures. Because of their in vitro selection and production, the technology of aptamers is emerging as a viable alternative for use in a broad range of applications including affinity chromatography, lateral flow devices and biosensors. Aptamers have been produced for several targets, including peptides, proteins, drugs, whole cells, and, recently, for mycotoxins, e.g. ochratoxin A (OTA) and fumonisin B1 (FB1). The DNA aptamer with high affinity and specificity to OTA was used as oligosorbent for the preparation of aptamer-based solid phase extraction (SPE) columns. The procedure for the preparation of SPE columns was standardised after evaluating the effect of different parameters, such as oligosorbent volume, column size and breakthrough volume. SPE columns packed with 300 µl oligosorbent (24 nmol aptamer) were successfully used for the clean-up of durum wheat extracts prior to OTA determination by high performance liquid chromatography (HPLC) and fluorescence detection (FLD) in unprocessed durum wheat. The SPE-columns showed a linearity of the dose-response curve in the range of 0.4-500 ng OTA. Average recoveries from wheat samples spiked at levels of 0.5-50 ng/g ranged from 74% to 88% (relative standard deviation <6%). The limit of quantification was 77 pg/g, far below the EU regulatory limits for OTA in unprocessed wheat (5.0 ng/g). Comparison of HPLC-FLD analyses of 33 naturally contaminated durum wheat samples after clean-up by aptamer-SPE or immunoaffinity (IMA) columns showed a good correlation (r = 0.990). Aptamer-SPE columns could be re-used up to five times without any loss of performance. These results provide evidence that this DNA aptamer is a promising material for the analysis of OTA in food and can be used to replace antibodies as binding reagents in diagnostic assays. Recently, six DNA sequences binding to FB1 have been selected in our laboratories after 18 SELEX rounds. In particular, a sequence (FB1 39) showed the highest binding efficiency towards FB1 with a dissociation constant (KD) in the nanomolar range. Further processing of the selected sequence are in progress to shorten the length and improve the binding affinity and to evaluate cross-reactivity towards other similar compounds. The final aptamer for FB1 will be evaluated for potential use in fumonisin detection systems. (literal)
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