We developed a simple and cheap assay for quantitatively detecting ochratoxin A (OTA) in wine. A DNA aptamer available in literature was used as recognition probe in its molecular beacon form, i.e., with a fluorescence-quenching pair at the stem ends. Our aptabeacon could adopt a conformation allowing OTA binding, causing a fluorescence rise due to the increased distance between fluorophore and quencher. We used real-time PCR equipment for capturing the signal. With this assay, under optimized conditions, the entire process can be completed within 1 h. In addition, the proposed system exhibited a good selectivity for OTA against other mycotoxins (ochratoxin B and aflatoxin M1) and limited interference from aflatoxin B1 and patulin. A wide linear detection range (0.2–2000 μM) was achieved, with LOD = 13 nM, r = 0.9952, and R2 = 0.9904. The aptabeacon was also applied to detect OTA in red wine spiked with the same dilution series. A linear correlation with a LOD = 19 nM, r = 0.9843, and R2 = 0.9708 was observed, with recoveries in the range 63%–105%. Intra- and inter-day assays confirmed its reproducibility. The proposed biosensor, although still being finalized, might significantly facilitate the quantitative detection of OTA in wine samples, thus improving their quality control from a food safety perspective.

Detection of ochratoxin a using molecular beacons and real-time PCR thermal cycler / Sanzani, S. M.; Reverberi, M.; Fanelli, C.; Ippolito, A.. - In: TOXINS. - ISSN 2072-6651. - 7:3(2015), pp. 812-820. [10.3390/toxins7030812]

Detection of ochratoxin a using molecular beacons and real-time PCR thermal cycler

Reverberi M.;Fanelli C.;
2015

Abstract

We developed a simple and cheap assay for quantitatively detecting ochratoxin A (OTA) in wine. A DNA aptamer available in literature was used as recognition probe in its molecular beacon form, i.e., with a fluorescence-quenching pair at the stem ends. Our aptabeacon could adopt a conformation allowing OTA binding, causing a fluorescence rise due to the increased distance between fluorophore and quencher. We used real-time PCR equipment for capturing the signal. With this assay, under optimized conditions, the entire process can be completed within 1 h. In addition, the proposed system exhibited a good selectivity for OTA against other mycotoxins (ochratoxin B and aflatoxin M1) and limited interference from aflatoxin B1 and patulin. A wide linear detection range (0.2–2000 μM) was achieved, with LOD = 13 nM, r = 0.9952, and R2 = 0.9904. The aptabeacon was also applied to detect OTA in red wine spiked with the same dilution series. A linear correlation with a LOD = 19 nM, r = 0.9843, and R2 = 0.9708 was observed, with recoveries in the range 63%–105%. Intra- and inter-day assays confirmed its reproducibility. The proposed biosensor, although still being finalized, might significantly facilitate the quantitative detection of OTA in wine samples, thus improving their quality control from a food safety perspective.
2015
aptabeacon; fluorescent dyes; food safety; OTA detection; real-time PCR thermal cycler
01 Pubblicazione su rivista::01a Articolo in rivista
Detection of ochratoxin a using molecular beacons and real-time PCR thermal cycler / Sanzani, S. M.; Reverberi, M.; Fanelli, C.; Ippolito, A.. - In: TOXINS. - ISSN 2072-6651. - 7:3(2015), pp. 812-820. [10.3390/toxins7030812]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11573/1685269
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