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Toxins 2015, 7(3), 812-820; doi:10.3390/toxins7030812

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

1
Department of Soil, Plant, and Food Sciences, University of Bari Aldo Moro, Via G. Amendola 165/A, 70126 Bari, Italy
2
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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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. 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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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

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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