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

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. Graphical abstractSchematic 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.