摘要
核酸扩增反应能够在短时间内将特定DNA模板的数量增加106~109倍,显著提高微量核酸检测的灵敏度,但同时扩增产物的遗漏极易造成交叉污染,影响后续检测。扩增反应通过添加荧光染料实现的实时闭管检测,不仅可以避免反应结束后开管操作所导致的交叉污染,还可以通过反应动力学对样品中模板的起始拷贝数定量,以及通过熔解曲线分析鉴别多种靶标来源、进行基因突变扫描和基因分型检测。目前常用的商业化实时检测荧光染料,如SYBR Green I、Eva Green等,其精确结构信息尚未披露,因此只能通过实验“试错法”选取合适染料,无法从根本上获知染料结构和分析性能之间的关系。本文将通过对目前常用的核酸实时检测荧光染料性质和应用进行总结,为其优化选择、设计和开发提供参考依据。
The nucleic acid amplification reaction can increase the number of specific DNA templates by 106-109 times at a short time.It can improve the detection sensitivity of trace nucleic acids significantly,but the residue of amplified products can easily cause cross-contamination and affect subsequent detection.The use of fluorescent dyes in the Real-time closed-tube detection for amplification reaction can not only avoid cross-contamination caused by open-tube operation after the reaction,but also quantify the initial copy number of the template in the sample by reaction kinetics,and identify various types of Target source,gene mutation scanning and genotyping testing through the melting curve analysis.At present,the precise structure of commonly used commercial fluorescent dyes for real-time detection,such as SYBR Green I,Eva green,etc.,has never been disclosed,so we can only obtain experience in selecting suitable dyes through Trial and Error,but cannot know the basic relationship between dye structure and analytical performance.This article will summarize the properties and applications of fluorescent dyes commonly used in real-time detection of nucleic acids,and provide a basis for their selection,design and development.
作者
邱婷
毛国斌
徐秦峰
QIU Ting;MAO Guobin;XU Qinfeng(Shanghai Institute of Organic Chemistry,Chinese Academy of Sciences,Shanghai 20032;CAS Key Laboratory of Quantitative Engineering Biology,Shenzhen Institutes of Advanced Technology,Chinese Academy of Sciences,Shenzhen,518055;School of Food Science and Engineering,Shanxi University of Science&Technology,Xi’an 710021)
出处
《分析科学学报》
CAS
CSCD
北大核心
2023年第4期479-486,共8页
Journal of Analytical Science
基金
中国科学院定量工程生物学重点实验室对外开放项目,国家自然科学基金项目(22074085)。
关键词
核酸扩增
实时检测
荧光染料
Nucleic acid amplification
Real-time detection
Fluorescence dyes
