基于核酸等温扩增与侧向层析试纸联用鉴定黑胡椒调味品的真实性

Identification of the authenticity of black pepper seasoning based on the combination of nucleic acid isothermal amplification and lateral chromatography testpaper

本研究以黑胡椒中木瓜籽掺假为代表性研究对象,开发了一种分子扩增与侧向流动层析(LFA)联用方法,通过整合重组酶聚合酶链式扩增反应(RPA)和LFA技术,实现了对黑胡椒样品中木瓜籽掺假的敏感快速检测。该方法的检出限(LOD)为0.1%,整个检测过程可以在40 min内完成,适用于现场测试,并成功用于鉴定香料黑胡椒的真实性。

In this study, an electrochemical sensor was constructed for the highly sensitive detection of microRNA-21 based on the tetrahedral DNA scaffold and G-quadruplex(G4) structure using a target-triggered enzyme-free strand displacement cyclic signal amplification strategy. A tetrahedral DNA scaffold hairpin probe was designed according to the sequence of the target(microRNA-21).In the presence of the target, it hybridized with the hairpin probe on the tetrahedral DNA scaffold by complementary pairing, which initiated the cyclic strand displacement reaction of three hairpin DNAs and formed a large number of "Y"-shaped DNA structures on the electrode surface. Each "Y"-shaped DNA structure has a G4 structure at both upper ends, which can bind with hemin to form a G4/hemin DNAzyme with horseradish peroxidase-like properties. It catalyzes the reaction between 3,3',5,5'-tetramethylbenzidine and hydrogen peroxide and generates significant current signals, which can be used for microRNA-21 detection. When the target was not present, the strand displacement reaction could not be initiated and thus the electrochemical signal was weak. The results showed there was a good linear relationship between the peak difference current and microRNA-21 concentration logarithm value in the range of 1.00 fmol/L-1.00 nmol/L, and the correlation equation was ΔI=45.04lgc+756.1, along with the correlation coefficient(R2) of 0.9987 and the detection limit of 17.92 amol/L. The biosensor is expected to be used in the clinical application of early diagnosis of miRNA-21-related tumors.