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

Toxins

Simona Marianna Sanzani¹  Massimo Reverberi²  Corrado Fanelli²  Antonio Ippolito¹ 

[1] Department of Soil, Plant, and Food Sciences, University of Bari Aldo Moro, Via G. Amendola 165/A, 70126 Bari, Italy; E-Mail:;Department of Environmental Biology, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy; E-Mails:

关键词: OTA detection;    fluorescent dyes;    aptabeacon;    Real-time PCR thermal cycler;    food safety;   

DOI  :  10.3390/toxins7030812

来源: mdpi

【 摘 要 】

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.

【 授权许可】

CC BY   
© 2015 by the authors; licensee MDPI, Basel, Switzerland.