title: Detection of ochratoxin A using molecular beacons and real-time PCR thermal cycler.
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.
authors.affiliation: Department of Soil, Plant, and Food Sciences, University of Bari Aldo Moro, Via G. Amendola 165/A, 70126 Bari, Italy. simonamarianna.sanzani@uniba.it.
authors.forename: Simona Marianna
authors.initials: SM
authors.lastname: Sanzani
authors.affiliation: Department of Environmental Biology, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy. massimo.reverberi@uniroma1.it.
authors.forename: Massimo
authors.initials: M
authors.lastname: Reverberi
authors.affiliation: Department of Environmental Biology, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy. corrado.fanelli@uniroma1.it.
authors.forename: Corrado
authors.initials: C
authors.lastname: Fanelli
authors.affiliation: Department of Soil, Plant, and Food Sciences, University of Bari Aldo Moro, Via G. Amendola 165/A, 70126 Bari, Italy. antonio.ippolito@uniba.it.
authors.forename: Antonio
authors.initials: A
authors.lastname: Ippolito
mesh_terms: D016607:Aflatoxin M1 / Q000032:analysis; D001231:Aspergillus flavus / Q000737:chemistry; D001235:Aspergillus ochraceus / Q000737:chemistry; D015374:Biosensing Techniques; D005456:Fluorescent Dyes; D005506:Food Contamination / Q000032:analysis; D005516:Food Microbiology; D059022:Food Safety; D057230:Limit of Detection; D009793:Ochratoxins / Q000032:analysis*; D010407:Penicillium / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014920:Wine / Q000032:analysis* / Q000382:microbiology
chemical_list: D005456:Fluorescent Dyes; D009793:Ochratoxins; C025589:ochratoxin A; D016607:Aflatoxin M1; C045354:ochratoxin B