高等学校化学学报 ›› 2016, Vol. 37 ›› Issue (1): 12-18.doi: 10.7503/cjcu20150524

• 分析化学 • 上一篇    下一篇

基于链置换循环无标记检测端粒酶RNA的荧光法

张霞菲, 成锐, 时志路, 金燕()   

  1. 陕西师范大学化学化工学院, 陕西省生命分析重点实验室, 陕西 710062
  • 收稿日期:2015-07-08 出版日期:2016-01-10 发布日期:2015-12-20
  • 基金资助:
    国家自然科学基金(批准号: 21075079, 21375086)、 陕西省创新团队研究计划项目(批准号: 2014KCT-28)、 陕西师范大学中央高校基金项目(批准号: GK261001097)和长江学者高校创新团队项目(批准号: IRT-14R33)资助

Label-free Fluorescence Assay of Telomerase RNA Based on Strand Displacement Amplification

ZHANG Xiafei, CHENG Rui, SHI Zhilu, JIN Yan*()   

  1. Analytical Chemistry for Life Science of Shaanxi Province, School of Chemistry and Chemical Engineering, Shaanxi Normal University, Xi’an 710062, China
  • Received:2015-07-08 Online:2016-01-10 Published:2015-12-20
  • Contact: JIN Yan E-mail:jinyan@snnu.edu.cn
  • Supported by:
    † Supported by the National Natural Science Foundation of China(Nos.21075079, 21375086), the Program for Innovative Research Team in Shaanxi Province, China(No.2014KCT-28), the Fundamental Research Funds for the Central Universities, China(No.GK261001097) and the Program for Changjiang Scholars and Innovative Research Team in University, China(No.IRT-14R33)

摘要:

以氧化石墨烯(GO)作为DNA载体和荧光猝灭剂, SYBR Green Ⅰ(SGⅠ)为荧光信号探针, 发夹核酸探针为分子识别探针, 基于目标物启动的发夹核酸探针链置换循环反应, 建立了一种利用荧光共振能量转移和链置换循环放大技术检测端粒酶RNA(hTR)的荧光新方法. 发夹核酸探针hpDNA1和hpDNA2吸附在GO表面, 嵌插在发夹DNA探针茎部的SGⅠ的荧光信号被GO猝灭. 当人工合成的目标物(T1)存在时, T1与hpDNA1杂交打开hpDNA1的茎-环结构而引发hpDNA2与T1之间的链置换循环反应, 由此累积产生大量的hpDNA1/hpDNA2杂交双链. 刚性的双链DNA脱离GO表面, 导致所嵌插的SGⅠ产生较强的荧光信号. 基于荧光信号的变化, 可定量检测0.2~50 nmol/L的T1, 检出限为90 pmol/L. 该方法为端粒酶RNA检测提供了一种高灵敏、 高特异性且无需标记的荧光新途径.

关键词: 端粒酶RNA, 链置换循环, 荧光共振能量转移, 无标记

Abstract:

A novel fluorescence method was developed for detecting telomerase RNA(hTR) based on the fluorescence resonance energy transfer(FRET) and strand displacement amplification(SDA) technique. Graphene oxide(GO) served as DNA carrier and fluorescence quencher. SYBR Green Ⅰ(SGⅠ) and hairpin DNAs(hpDNA) are fluorescence probe and molecular recognition probes, respectively. The fluorescence of SGⅠ that intercalated into the stem of hairpin DNAs was quenched when hpDNA1 and hpDNA2 were adsorbed onto the surface of GO. In the presence of T1, the hybridization reaction between hpDNA1 and T1 opened the hairpin structure of hpDNA1 to trigger the SDA reaction between hpDNA2 and T1, leading to an accumulation of hpDNA1/hpDNA2 hybrids. The rigid dsDNA desorbed from GO surface to restore the fluorescence of SGⅠ. Based on the change in fluorescence intensity, T1 can be quantitatively detected from 0.2 nmol/L to 50 nmol/L, with a detection limit of 90 pmol/L. Therefore, it offers a label-free, highly sensitive and specific fluorescence strategy for detection of hTR.

Key words: Telomerase RNA, Strand displacement amplification, Fluorescence resonance energy transfer, Label-free

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