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Toxins 2015, 7(3), 812-820;

Detection of Ochratoxin a Using Molecular Beacons and Real-Time PCR Thermal Cycler

Department of Soil, Plant, and Food Sciences, University of Bari Aldo Moro, Via G. Amendola 165/A, 70126 Bari, Italy
Department of Environmental Biology, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
Author to whom correspondence should be addressed.
Academic Editor: Richard A. Manderville
Received: 12 December 2014 / Revised: 26 February 2015 / Accepted: 2 March 2015 / Published: 9 March 2015
(This article belongs to the Collection Ochratoxins-Collection)
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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. View Full-Text
Keywords: OTA detection; fluorescent dyes; aptabeacon; Real-time PCR thermal cycler; food safety OTA detection; fluorescent dyes; aptabeacon; Real-time PCR thermal cycler; food safety

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Sanzani, S.M.; Reverberi, M.; Fanelli, C.; Ippolito, A. Detection of Ochratoxin a Using Molecular Beacons and Real-Time PCR Thermal Cycler. Toxins 2015, 7, 812-820.

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