Guo Wei, Yang Haoyu, Zhang Yunzhe, Wu Hao, Lu Xin, Tan Jianxin, Zhang Wei
College of Food Science and Technology, Hebei Agricultural University, Baoding 071000, China.
Hebei Province Feed Microorganism Technology Innovation Center, Baoding 071000, China.
Foods. 2022 Aug 13;11(16):2443. doi: 10.3390/foods11162443.
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 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, with the lowest limit of detection of 0.01 ng mL. 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 96.1~100%. The results suggest that the developed sensor has a promising potential application for the ultrasensitive detection of contaminants in food.
由于赭曲霉毒素A毒性很高，因此迫切需要开发方便、可靠、超灵敏且特异的方法来测定食品安全中的赭曲霉毒素A。在本研究中，构建了一种将实时荧光与链置换扩增（SDA）相结合的超灵敏、无标记荧光适体传感器，用于测定OTA。在存在OTA的情况下，OTA适体与OTA结合，从而打开发夹结构。然后，SDA引物特异性结合发夹茎，将其作为模板用于后续扩增。在DNA聚合酶和切口内切核酸酶的作用下启动SDA扩增。扩增产物（单链DNA）用SYBR Green II染色，并用实时荧光进行检测。该方法在0.01-50 ng/mL范围内具有良好的线性，最低检测限为0.01 ng/mL。此外，荧光适体传感器具有出色的特异性和重现性。此外，该传感器在小麦和燕麦样品的人工加标检测中表现出优异的分析性能，回收率为96.1%~100%。结果表明，所开发的传感器在食品中污染物的超灵敏检测方面具有广阔的潜在应用前景。
