分析测试学报2025，Vol.44Issue(11)：2346-2353,8.DOI:10.12452/j.fxcsxb.25011324
等温链置换扩增反应/CRISPR-Cas12a系统结合反向荧光增强试纸条检测唾液中microRNA-31的研究
Isothermal Strand Displacement Amplification Reaction/CRISPR-Cas12a System for Detection of MicroRNA-31 in Saliva Samples by Combining with Reverse Fluorescence Enhancement Lateral Flow Test Strip
摘要
Abstract
A microRNA-31(miRNA-31)amplifying detection signals method based on nucleic acid isothermal strand displacement amplification(ISDA)and CRISPR-Casl2a system were established and achieved visualizing detection by combining with reverse fluorescence enhancement lateral flow strip(rLFTS)platform.Under optimizing the primer toehold design and reaction buffer of ISDA reac-tion,miRNA-31 signal was efficiently amplified to output copious double stranded DNA,which ac-tivated CRISPR-Cas12a trans-cleavage activity to digest single-stranded reporter molecules and led to gold nanoparticles couldn't be captured on the test lines by sandwich method to quench Cy5 fluores-cence,causing corresponding quantitative fluorescence signals for improving miRNA-31 detection sensitivity.The results showed that the detection limit of 57.9 amol/L and recoveries of 91.6％-1 11％for detection miRNA-31 in saliva samples by the method.Moreover,the quantitative results had good consistency with qRT-PCR method in a much faster detection time(90 min).Given its simplici-ty,sensitivity and rapid,ISDA-Cas12a-rLFTS could meet the demand for ultra-sensitive detection of miRNA-31 in saliva samples.关键词
等温链置换扩增/CRISPR-Cas12a/反向荧光增强试纸条/miRNA-31/核酸检测Key words
isothermal strand displacement amplification/CRISPR-Cas12a/reverse fluorescence enhancement lateral flow test strip/miRNA-31/nucleic acid detection分类
化学引用本文复制引用
王佳讯,徐一,赵爽,宫晓群..等温链置换扩增反应/CRISPR-Cas12a系统结合反向荧光增强试纸条检测唾液中microRNA-31的研究[J].分析测试学报,2025,44(11):2346-2353,8.基金项目
国家自然科学基金(U2233206) （U2233206）
