基于核酸适配体的荧光传感器检测赭曲霉毒素A

Detection of ochratoxin A by fluorescence sensor based on aptamer

为了实现对食品中赭曲霉毒素A的快速检测,利用带有羧基荧光基团(FAM)的核酸适配体(Apt-FAM)与带有黑洞猝灭基团(BHQ1)的互补链cDNA-BHQ1,构建了简单、快速检测食品中赭曲霉毒素A(ochratoxin A,OTA)的荧光传感器。研究结果表明:当OTA不存在时,Apt-FAM通过碱基互补配对与cDNA-BHQ1结合,荧光基团FAM靠近猝灭基团BHQ1,通过荧光共振能量转移作用抑制荧光基团FAM的信号;当OTA存在时,OTA与Apt-FAM结合形成G-四链体单链结构,G-四链体远离cDNA-BHQ1,荧光信号增强。通过试验条件的优化,选择最优检测条件:互补链为cDNA1-BHQ1、孵育时间为60 min、cDNA与Apt用量比为1∶1、反应体系的pH值为7.4。在最优检测条件下,传感器检测OTA的线性范围为0.5~100 ng/mL,线性方程为y=31.934 98+41.714 94lg(C_(OTA)),R^(2)=0.997,最低检出限为0.13 ng/mL。利用该传感器检测玉米粉和葡萄酒样品中的OTA,平均回收率分别为100.0%~102.0%和98.0%~102.2%。研究构建的荧光传感器成功用于快速检测食品中OTA,为快速检测食品中真菌毒素提供了参考。

Ochratoxin A is a kind of common fungal toxin that mainly contaminates food crops and their products.It is stable and not easily degraded when residing in food.Toxicological studies have shown that OTA is harmful to human and animal health since it has strong hepatotoxicity,nephrotoxicity,neurotoxicity,and teratogenicity.To ensure food safety and human health,it is required to establish simple and rapid detection methods.In this experiment,a fluorescent sensor based on DNA aptamer was constructed for the rapid detection of OTA in food.A simple and rapid fluorescent sensor for OTA detection was constructed by using the aptamer with carboxyl fluorescein(FAM)and the complementary chain with black hole quenching group(BHQ1).When OTA is absent,Apt-FAM binds to cDNA-BHQ1 by base complementary pairing,the fluorescent group FAM is close to the quenching group BHQ1,and the signal of fluorescent group FAM is suppressed by the effect of fluorescence resonance energy transfer.When OTA is present,OTA binds to Apt-FAM to form G-quadruplex,which keeps away from cDNA-BHQ1,and the fluorescent signal is restored.By controlling the distance between the FAM fluorophore and the quenching group BHQ1,the intensity of the fluorescence signal is changed,thereby realizing the detection of OTA.In order to optimize the detection performance of the sensor,the number of bases and complementary positions of complementary strands,the total reaction time,the ratio of cDNA to Apt dosage,and the pH of the reaction system were optimized.Through the optimization of experimental conditions,the optimal complementary strand was cDNA1-BHQ1,the optimal reaction time was 60 min,the optimal dosage ratio of cDNA to Apt was 1∶1,and the pH value of the optimal reaction system was 7.4.In the range of OTA concentration from 0.5 ng/mL to 100 ng/mL,the relative fluorescence intensity and the OTA concentration were logarithmic,showing a good linear relationship.The linear equation is y=31.93498+41.71494 lg(C_(OTA)),and the correlation coefficient R^(2)=0.997,with a detection limit of 0.13 ng/mL.The experimental results showed that in the evaluation results of anti-interference performance of OTA and other toxins,the fluorescence sensor had good specificity for detecting OTA.The sensor also had good reproducibility.The prepared fluorescent sensor was used for spiked detection of corn samples and wine samples,and the average recoveries were 100.0%-102.0%and 98.0%-102.2%respectively,indicating that the sensor can be used for the detection of OTA in actual samples.In conclusion,a simple and fast fluorescent aptamer sensor for high sensitivity and effective detection of OTA has been successfully designed,which is of great significance for ensuring food safety.