基于寡核苷酸链信号放大快速检测谷物中赭曲霉毒素A

Rapid Detection of Ochratoxin A in Cereals Based on Oligonucleotide Chain Signal Amplification

该研究以柠檬酸钠还原法制备粒径均一的金纳米粒子(gold nanoparticles,AuNPs),并将AuNPs与赭曲霉毒素A(ochratoxin A,OTA)抗体通过静电吸附作用偶联,再与6-羧基荧光素(6-carboxyfluorescein,6-FAM)修饰的寡核苷酸链(single stranded DNA,ssDNA)通过金硫键偶联,制备出荧光探针,利用AuNPs近距离淬灭荧光基团信号作用和1,4-二巯基苏糖醇(dithiothreitol,DTT)解离寡核苷酸链作用,建立基于寡核苷酸链信号放大快速检测谷物中OTA的方法。经过优化试验条件,该方法的检测限为0.0044μg/L,特异性良好。使用该方法测定玉米、大米、燕麦3种谷物中的OTA含量,添加回收率为91.2%~102.3%,变异系数小于10%,检测结果与超高效液相色谱串联质谱检测结果接近,证明该方法能够用于检测谷物样品中OTA的含量。

In this study,gold nanoparticles(AuNPs)with uniform particle size were prepared by the sodium citrate reduction method.The fluorescence probe was prepared by coupling AuNPs with ochratoxin A(OTA)antibody through electrostatic adsorption,and the resultant product was coupled with 6-carboxyfluorescein(6-FAM)-modified oligonucleotide chain(ssDNA)through gold sulfur bond.AuNPs were used to quench the fluorescence group signal in a close range and 1,4-dithiothreitol(DTT)was used to dissociate the oligonucleotide chain to establish a method for rapid detection of OTA in cereals based on oligonucleotide chain signal amplification.The limit of detection of the method was 0.0044μg/L with good specificity under the optimized conditions.The OTA content in corn,rice,and oat was determined by this method.The results showed that the added recovery rate was in the range of 91.2%-102.3%,and the coefficient of variation was less than 10%.The detection results were close to those of ultra-performance liquid chromatography-tandem mass spectrometry(UPLC-MS/MS),which proved that the method could be used to detect the content of OTA in cereals.