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

Sanzani SM, Reverberi M, Fanelli C, Ippolito A - Toxins (Basel) (2015)

Bottom Line: 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%.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 Article: PubMed Central - PubMed

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.

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.

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Related in: MedlinePlus

Selectivity of the aptameric assay system toward ochratoxin A (OTA) against other mycotoxins (OTB, ochratoxin B; AFB1, aflatoxin B1; AFM1, aflatoxin M1; PAT, patulin) at the same concentration (2 µM). The blank signal (aptabeacon in absence of OTA) was subtracted to all samples. (A) Instrument screen; (B) toxins’ peak areas. Each bar represents the average value of three independent experiments (with three technical replicates each) with error bars indicating standard error of the mean (SEM). Significant differences (p ≤ 0.05) were identified with the Duncan’s Multiple Range Test: bars with different letters are significantly different.
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toxins-07-00812-f002: Selectivity of the aptameric assay system toward ochratoxin A (OTA) against other mycotoxins (OTB, ochratoxin B; AFB1, aflatoxin B1; AFM1, aflatoxin M1; PAT, patulin) at the same concentration (2 µM). The blank signal (aptabeacon in absence of OTA) was subtracted to all samples. (A) Instrument screen; (B) toxins’ peak areas. Each bar represents the average value of three independent experiments (with three technical replicates each) with error bars indicating standard error of the mean (SEM). Significant differences (p ≤ 0.05) were identified with the Duncan’s Multiple Range Test: bars with different letters are significantly different.

Mentions: Since the selectivity is another important issue for practical implementation of OTA detectors, we compared the change of fluorescent intensity induced by other relevant mycotoxins (OTB, AFB1, AFM1, and PAT) with a potential combination ability with OTA aptamer [8]. As shown in Figure 2, at the same concentration, the aptabeacon exhibited evident different response signals to the mycotoxins. The response signals to OTB (OTA structural analogue) and AFM1 in term of peak area were on average 67–88% lower than that to OTA, whereas those of AFB1 or PAT curves were up to 41% lower. Aflatoxins have a different product range [10] and patulin, although potentially present on grapes [11] disappears rapidly during fermentation to essentially a zero level [12]. These findings, together with the difference in affinity between OTA and OTB with the aptamer, were considered even in previous similar investigations sufficient for practical application [7].


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

Sanzani SM, Reverberi M, Fanelli C, Ippolito A - Toxins (Basel) (2015)

Selectivity of the aptameric assay system toward ochratoxin A (OTA) against other mycotoxins (OTB, ochratoxin B; AFB1, aflatoxin B1; AFM1, aflatoxin M1; PAT, patulin) at the same concentration (2 µM). The blank signal (aptabeacon in absence of OTA) was subtracted to all samples. (A) Instrument screen; (B) toxins’ peak areas. Each bar represents the average value of three independent experiments (with three technical replicates each) with error bars indicating standard error of the mean (SEM). Significant differences (p ≤ 0.05) were identified with the Duncan’s Multiple Range Test: bars with different letters are significantly different.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4379526&req=5

toxins-07-00812-f002: Selectivity of the aptameric assay system toward ochratoxin A (OTA) against other mycotoxins (OTB, ochratoxin B; AFB1, aflatoxin B1; AFM1, aflatoxin M1; PAT, patulin) at the same concentration (2 µM). The blank signal (aptabeacon in absence of OTA) was subtracted to all samples. (A) Instrument screen; (B) toxins’ peak areas. Each bar represents the average value of three independent experiments (with three technical replicates each) with error bars indicating standard error of the mean (SEM). Significant differences (p ≤ 0.05) were identified with the Duncan’s Multiple Range Test: bars with different letters are significantly different.
Mentions: Since the selectivity is another important issue for practical implementation of OTA detectors, we compared the change of fluorescent intensity induced by other relevant mycotoxins (OTB, AFB1, AFM1, and PAT) with a potential combination ability with OTA aptamer [8]. As shown in Figure 2, at the same concentration, the aptabeacon exhibited evident different response signals to the mycotoxins. The response signals to OTB (OTA structural analogue) and AFM1 in term of peak area were on average 67–88% lower than that to OTA, whereas those of AFB1 or PAT curves were up to 41% lower. Aflatoxins have a different product range [10] and patulin, although potentially present on grapes [11] disappears rapidly during fermentation to essentially a zero level [12]. These findings, together with the difference in affinity between OTA and OTB with the aptamer, were considered even in previous similar investigations sufficient for practical application [7].

Bottom Line: 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%.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 Article: PubMed Central - PubMed

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.

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.

Show MeSH
Related in: MedlinePlus