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核酸探针杂交及酶联显色方法定量检测微小RNA-21
作者:于天晓  许红  韩彦洁  张瑶  王亚芳  张静蕊  李美芳  腰利云 
单位:石家庄市第四医院/石家庄市妇产医院输血科, 河北 石家庄 050000
关键词:微小RNA-21 检测方法 显色分析 
分类号:R-33
出版年·卷·期(页码):2019·47·第七期(805-807)
摘要:

目的:建立一种基于核酸探针杂交及酶联显色的高灵敏方法,实现对微小RNA-21(miRNA-21)的定性和定量检测。方法:将生物素标记的miRNA-21捕获探针通过生物素-亲和素系统固定在96孔板上,当靶miRNA-21出现在反应体系中时,能够与固定在96孔板上的捕获探针结合。利用辣根过氧化物酶标记的DNA检测探针的催化和信号放大作用,催化底物3,3',5,5'-四甲基联苯胺,使反应体系出现颜色变化,根据颜色变化对miRNA-21的浓度进行定性和定量分析。结果:通过考察方法的灵敏度、精密度、回收率可得该方法最低检测限可达0.81 pmol·L-1,相对标准偏差均在7.5%以下,回收率在78.9%~119.2%之间,结论:本研究建立了一种高通量显色分析方法,该方法能够高灵敏检测miRNA-21,且具有良好的灵敏度、重复性和回收率。

Objective:To established a high sensitivity method based on nucleic acid probe hybridization and enzyme-linked chromogenic for qualitative and quantitative detection of microRNA-21(miRNA-21). Methods:Biotin labelled miRNA-21 capture probe was immobilized on 96-well plate by biotin-streptavidin system. The capture probe could effectively bind to the target miRNA-21 which appeared in the reaction system. DNA detection probe labeled by horseradish peroxidase which had catalytic activity and signal amplification could catalytic substrate 3, 3', 5, 5'-tetramethylbenzidine to form a colorimetric reaction. Qualitative and quantitative analysis of the miRNA-21 concentration were performed based on the color change of reaction system. Result:By investigating the sensitivity,precision and recovery of the method, the lowest detection limit was 0.81 pmol·L-1, relative standard deviationwere less than 7.5%, and the recoveries were between 78.9%-119.2%.Conclusion:In this study, a sensitive and high throughput chromogenic method for detection of miRNA-21 is established. It has good sensitivity, repeatability and recovery.

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