基于桥接DNA的实时荧光定量PCR高灵敏检测沙丁胺醇

Highly Sensitive Detection of Salbutamol Based on Bridged DNA-based Real-time Fluorescent Quantitative PCR

建立一种基于免疫磁珠、桥接DNA与实时荧光定量聚合酶链式反应技术(quantitative real-time polymerase chain reaction,qPCR)相结合的高灵敏、特异性检测沙丁胺醇(salbutamol,SAL)的方法,通过抗体特异性识别两个邻位连接探针,在桥接DNA的桥接作用下将两个邻位探针连接形成全长扩增子,以此为模板进行qPCR实现信号放大。结果表明,方法检测SAL的线性范围为1.0×10^-2 ng/mL^1.0×10^3 ng/mL,检出限为1.2×10^-2 ng/mL,测定自来水及人工尿液样品中SAL的加标回收率在87.1%~111.7%之间。通过免疫磁珠提高检测的效率,通过桥联DNA提高特异性,通过变温扩增提高检测方法的灵敏度以及适用性,搭建高特异性、通用的检测平台,实现对沙丁胺醇的高灵敏检测。

A highly sensitive and specific method for the detection of salbutamol(SAL)based on immunomagnetic beads,bridged DNA and real-time quantitative PCR(qPCR)was established.Two proximity ligation assay probes were specifically identified by antibodies.Under the bridged DNA,two proximity ligation assay probes were ligated to form a full-length amplicon,and the products of the ligation reaction were quantified by using a quantitative real-time polymerase chain reaction.The results showed that the linear range of SAL detected by this method was 1.0×10^-2 ng/mL-1.0×10^3 ng/mL,the detection limit was 1.2×10^-2 ng/mL,and the spiked recovery of SAL in tap water and artificial urine samples was determined.The recovery rate was between 87.1% and 111.7%.The efficiency of detection was improved by immunomagnetic beads,the specificity of the bridged DNA was increased and the sensitivity and applicability of the detection method were improved by constant temperature amplification.A highly specific and universal detection platform was constructed to realize highly sensitive detection of salbutamol.