一种基于重组聚合酶扩增和纸基电化学发光的核酸检测方法  

A Nucleic Acid Detection Method Based on Recombinant Polymerase Amplification and Paper-Based Electrochemiluminescence

作　　者：夏舒 陈钰[2] 李昕 张海燕[2] 刘仲明[2] 王捷[2] XIA Shu;CHEN Yu;LI Xin;ZHANG Hai-yan;LIU Zhong-ming;WANG Jie(School of Biology and Biological Engineering South China University of Technology,Guangzhou 510006;Department of Medical Research,Guangzhou Genenral Hospital of Guangzhou Military Command,Guangzhou 510010)

机构地区：[1]华南理工大学生物科学与工程学院,广东广州510006 [2]广州军区广州总医院医学实验科,广东广州510010

出　　处：《分析科学学报》2018年第5期627-632,共6页Journal of Analytical Science

基　　金：国家青年自然科学基金(No.81401752);军队后勤科研计划(No.CGZ16J002);广州市科学(技术)研究专项(No.201607010121)

摘　　要：基于重组聚合酶扩增(RPA)技术和纸基电化学发光(ECL)技术,结合RPA高特异性、操作简便和ECL高灵敏度、快速等特点,建立了一种新型核酸检测方法。以乙型肝炎病毒(HBV)基因片段的质粒为模板,设计特异性引物,对上游引物进行三联吡啶钌标记,生物素标记下游引物,通过RPA反应形成三联吡啶钌和生物素双标记的扩增子,链霉亲和素磁微球分离扩增子,纸基ECL检测三联吡啶钌信号。考察了上游引物三联吡啶钌标记效果,标记率为74.62%。优化了RPA反应参数条件,表明于30℃扩增20min即达到最佳核酸扩增效果。在HBV基因质粒模板浓度为0.12~1 200ng/mL范围内,纸基ECL响应峰面积与其浓度对数值呈现良好的线性关系,检出限(S/N>3)为1.2pg/mL。本方法可为在条件有限的现场进行核酸的快速检测提供实验依据。Based on recombinant polymerase amplification（RPA）technology and paper-based electrochemiluminescence（ECL）technology,a novel nucleic acid detection method was established.Using the plasmid including hepatitis B virus（HBV）gene fragment as a template,specific primers were designed and the upstream primers were labeled with tripyridine ruthenium,and biotin was used to combine with the downstream primers.Tripyridine ruthenium and biotin double-labeled amplificon was generated after RPA,then streptavidin magnetic microspheres were used to separate amplicon from the solution,and the signal emitted by tripyridine ruthenium was detected by paper-based ECL.The effect of tripyridine ruthenium labeling upstream primer was investigated,and the labeling rate was 74.62%.After optimizing relevant RPA parameters,results suggested that amplification of nucleic acid at 20℃for 20 min achieved the optimal performance.In the range of 0.12-1 200 ng/mL of HBV gene plasmid template concentration,the linear relationship between response area of paper-based ECL and logarithm of concentration was good,and the detection limit was1.2 pg/mL（S/N〉3）.This method can provide experimental evidence for rapid detection of nucleic acids in the areas with limited conditions.

关 键 词：重组聚合酶扩增 电化学发光 核酸检测 纸基 

分 类 号：O657.39[理学—分析化学]