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A fluorometric aptamer-based assay for ochratoxin A by using exonuclease III-assisted recycling amplification

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Abstract

A fluorometric assay is described for ochratoxin A (OTA) using an aptamer. The method is based on exonuclease-assisted recycling amplification. The OTA-binding aptamer partially hybridizes with complementary DNA (cDNA) that is released when the aptamer recognizes OTA. Then, cDNA hybridizes with a specifically designed hairpin DNA. Next, short ssDNA and cDNA are, respectively, released by exonuclease III catalyzed hydrolysis of the dsDNA. The cDNA induces the next ring opening and digestion. The short ssDNA captures the sDNA that is labeled with fluorescent FAM and is absorbed on graphene oxide (GO). The green fluorescence of the sDNA/GO system is quenched but is recovered if the sDNA is released from GO. This assay is high sensitive, works in the 5 nM to 200 nM OTA concentration range and has a 0.96 nM lower detection limit. It was applied to the quantitation of OTA in spiked wine and coffee samples.

Schematic of a fluorometric assay based on exonuclease-assisted recycling amplification for quantitative monitoring of OTA without the need of sample separation and multiple washing steps.

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Acknowledgements

This work was supported by the Fundamental Research Funds for the Central Universities (No. GK201802012) and Shaanxi Science Technology Plan Projects (No. 2017NY-121).

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Authors and Affiliations

  1. Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, College of Food Engineering and Nutritional Science, Shaanxi Normal University, Xi’an, 710119, China

    Mei Liu, Xuanyi Li & Zongqi Yang

  2. Key Laboratory of Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry & Chemical Engineering, Shaanxi Normal University, Xi’an, 710119, China

    Baoxin Li & Jianxiu Du

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  1. Mei Liu
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Liu, M., Li, X., Li, B. et al. A fluorometric aptamer-based assay for ochratoxin A by using exonuclease III-assisted recycling amplification. Microchim Acta 187, 46 (2020). https://doi.org/10.1007/s00604-019-3992-6

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