A Novel Fluorescent Aptasensor Based on Real-Time Fluorescence and Strand Displacement Amplification for the Detection of Ochratoxin A

It is urgently necessary to develop convenient, reliable, ultrasensitive and specific methods of ochratoxin A determination in food safety owing to its high toxicity. In the present study, an ultrasensitive and labeled-free fluorescent aptamer sensor combining real-time fluorescence with strand displacement amplification (SDA) was fabricated for the determination of OTA. In the presence of OTA, the OTA-aptamer combines with OTA, thus opening hairpins. Then, SDA primers specifically bind to the hairpin stem, which is used for subsequent amplification as a template. SDA amplification is initiated under the action of Bst DNA polymerase and nicking endonuclease. The amplified products (ssDNA) are dyed with SYBR Green II and detected with real-time fluorescence. The method has good linearity in the range of 0.01-50 ng mL(-1), with the lowest limit of detection of 0.01 ng mL(-1). Additionally, the fluorescent aptamer sensor shows outstanding specificity and reproducibility. Furthermore, the sensor shows excellent analytical performance in the artificial labeled detection of wheat and oat samples, with a recovery rate of 96.1 similar to 100%. The results suggest that the developed sensor has a promising potential application for the ultrasensitive detection of contaminants in food.