核酸适配体识别-荧光法检测赭曲霉素A

摘要

建立了核酸适配体识别-荧光探针技术检测赭曲霉素A(OTA)的新方法.基于微孔板上固定的核酸适配子与目标物质OTA结合时构象发生变化,导致预先与其互补杂交的FAM标记短链DNA解离,引起荧光信号发生变化,据此可实现对OTA的定量检测.当微孔板包被亲和素浓度为25 mg/L、适配子浓度为50 nmol/L,FAM标记互补短链DNA用量为150 nmol/L,OTA结合缓冲液为10 mmol/L HEPES(含120 mmol/L NaCl、5 mmol/L KCl、20 mmol/L MgCl2、20 mmol/L CaCl2,pH 7.0),45 ℃反应40 min时,可获得方法的最佳分析结果.在最佳实验条件下,对OTA的检测线性范围为2.0 ×10-8～1.0 ×10-5 g/L;检出限为1×10-8 g/L;相对标准偏差为2.6%(1×10-6 g/L,n＝11).本方法选择性良好,操作简单,已成功应用于实际样品中OTA的检测.%A novel analytical method for the detection of Ochratoxin A(OTA) was established based on the aptamer recognition and fluorescent probe technology. The method was developed according to the fact that when the immobilized aptamer bond to the target OTA, it can induce the conformation change of aptamer and result in the dissociation of the carboxy-fluorescein(FAM)-tagged complementary DNA chain from aptamer, and finally lead to the fluorescent signals change. Based on it, OTA can be quantified. All the condition factors affecting the performance of the present method were investigated. The results show if the avidin concentration coated on the microplate is 25 mmg/L, the aptamer concentration is 50 nmol/L, the concentration of FAM-tagged complementary DNA chain is 150 nmol/L, 10 mmol/L n-2-hydroxyethyl-piperazine-n-ethane sulfonic acid(HEPES) pH 7.0(contain 120 mmol/L NaCl, 5 mmol/L KCl, 20 mmol/L MgC12, 20 mmol/L CaCl2) was chosen as the binding buffer solution and the binding reaction was conducted at 45 ℃ for 40 min, the optimal analytical performance can be achieved. Under the optimal conditions, the linear range for the OTA concentration detection is 2.0 × 10- 11-1.0 × 10-8 g/mL with a detection limit of 1 × 10-11 g/mL. The RSD is 2.6％ for 11 parallel measurements of 1 × 10-9 g/mL OTA. Meanwhile, the present method is highly selective for OTA and easy to be operated. It has been successfully applied to measure OTA content in real samples.