核酸侵入反应偶联纳米金探针杂交显色技术可视化检测乙醛脱氢酶2基因多态性位点

引用本文: 盛楠, 岳慧杰, 逄淑云, 齐谢敏, 张晏洁, 吴燕子, 宋沁馨, 邹秉杰, 周国华. 核酸侵入反应偶联纳米金探针杂交显色技术可视化检测乙醛脱氢酶2基因多态性位点[J]. 分析化学, 2021, 49(1): 42-49. doi: 10.19756/j.issn.0253-3820.201234 shu

Citation: SHENG Nan, YUE Hui-Jie, PANG Shu-Yun, QI Xie-Min, ZHANG Yan-Jie, WU Yan-Zi, SONG Qin-Xin, ZOU Bing-Jie, ZHOU Guo-Hua. Visualized Detection of Aldehyde Dehydrogenase 2 Gene Polymorphism by Serial Invasive Reaction Coupled with Gold Nanoparticle Probe Assembling[J]. Chinese Journal of Analytical Chemistry, 2021, 49(1): 42-49. doi: 10.19756/j.issn.0253-3820.201234 shu

核酸侵入反应偶联纳米金探针杂交显色技术可视化检测乙醛脱氢酶2基因多态性位点

盛楠 ^1, ,
岳慧杰 ^1, ,
逄淑云 ^1, ,
齐谢敏 ^1, ,
张晏洁 ^1, ,
吴燕子 ^1, ,
宋沁馨 ^1,2, ,
邹秉杰 ^{1,3,
,} ,
周国华 ^{1,3,4,
,}

1.
东部战区总医院药理科, 南京 210002;
2.
中国药科大学, 药物质量与安全预警教育部重点实验室, 南京 210009;
3.
南京大学生命分析化学国家重点实验室, 南京 210093;
4.
南方医科大学第一临床医学院, 南京 210002

通讯作者: 邹秉杰,E-mail:zbj523@163.com; 周国华,E-mail:ghzhou@nju.edu.cn

基金项目:
国家自然科学基金项目（Nos.61871403，81703474，81673390）、江苏省青年医学人才项目（No.QNRC2016889）和江苏省自然科学基金项目（No.BK20180292）资助

摘要: 传统的乙醛脱氢酶2（Aldehyde dehydrogenase 2，ALDH2）基因多态性位点（rs671）分型手段包括电泳、测序和荧光探针法，但在实际应用中存在步骤繁琐或检测成本高等问题。本研究基于聚合酶链式反应（Polymerase chain reaction，PCR），将核酸侵入反应和纳米金探针（Gold nanoparticle probe，GNP）杂交显色技术偶联，建立了操作简便的ALDH2基因rs671位点可视化检测方法。在反应体系中，首先对样本DNA进行PCR扩增，再由级联核酸侵入反应识别等位基因，随后加入两种GNP与溶液中的发夹探针杂交显色。当样本DNA包含靶标等位基因时，体系中的发夹探针会在核酸侵入反应阶段被核酸内切酶1（Flap endonuclease 1，FEN1）切割裂解成两部分，这两部分序列分别与两种GNP杂交并分散在溶液中，最终溶液为红色；无靶标等位基因时，体系中的发夹探针保持完整结构，同时与两种GNP杂交，溶液中的GNP聚集，沉淀至管底，最终溶液为无色。因此，根据反应最终溶液的颜色可直接判断基因型。在最优条件下，本方法成功检测了低至80 pg的基因组DNA，并能准确区分3种基因型，表明本方法具有极高的灵敏度和特异性。将本方法用于临床样本ALDH2基因的rs671位点检测，207例样本可视化检测结果与焦磷酸测序分型结果完全一致，表明本方法在精准医学相关的单核苷酸多态性（Single nucleotide polymorphisms，SNP）分型检测方面有潜在的应用前景。

关键词:

English

Visualized Detection of Aldehyde Dehydrogenase 2 Gene Polymorphism by Serial Invasive Reaction Coupled with Gold Nanoparticle Probe Assembling

1.
Department of Pharmacology, Jinling Hospital, Medical School of Nanjing University, Nanjing 210002, China;
2.
Key Laboratory of Drug Quality Control and Pharmacovigilance of Ministry of Education, China Pharmaceutical University, Nanjing 210009, China;
3.
Department of Chemistry, State Key Laboratory of Analytical Chemistry for Life Science, Nanjing University, Nanjing 210093, China;
4.
The First School of Clinical Medicine, Southern Medical University, Nanjing 210002, China

Corresponding author: ZOU Bing-Jie, ; ZHOU Guo-Hua,

Received Date: 26 April 2020
Revised Date: 27 October 2020
Fund Project:
Supported by the National Natural Science Foundation of China (Nos.61871403, 81703474, 81673390), the Jiangsu Provincial Medical Youth Talent Program (No.QNRC2016889) and the Natural Science Foundation of Jiangsu Province (No.BK20180292).

Abstract: Conventional assays for identifying polymorphism of aldehyde dehydrogenase 2 (ALDH2) gene (rs671) are based on electrophoresis, fluorescence-labeled probes or sequencing, which are laborious and expensive. Herein, a colorimetric method was proposed for detection of ALDH2 genotype by PCR-based serial invasive reaction coupled with gold nanoparticle probe (GNP) assembling, in which the target DNAs were firstly amplified by PCR, and then the alleles on the amplicons were identified by serial invasive reaction, and finally the genotyping results could be read out with naked eyes by observing the color changes caused by gold nanoparticle assembling. If the allele-specific invasive probe matched the target sequence, the hairpin probe could be cleaved into two parts by Flap endonuclease 1 (FEN1). As a result, the GNPs could not be aggregated by the hairpin probe, keeping the solution red. Otherwise, the intact hairpin probe caused the aggregation of GNPs, leading to the color change from red to purple or clear. Therefore, the genotyping result could be read out with naked eyes by observing the color changes of the reaction. As low as 80 pg of genomic DNA could be accurately genotyped, and the genotyping results of 207 clinical samples were consisted with that of pyrosequencing, indicating that the proposed method has great potential in genotyping of personalized medicine-related single nucleotide polymorphism.

Key words:

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核酸侵入反应偶联纳米金探针杂交显色技术可视化检测乙醛脱氢酶2基因多态性位点

通讯作者: 邹秉杰,E-mail:zbj523@163.com; 周国华,E-mail:ghzhou@nju.edu.cn

English

Visualized Detection of Aldehyde Dehydrogenase 2 Gene Polymorphism by Serial Invasive Reaction Coupled with Gold Nanoparticle Probe Assembling

Corresponding author: ZOU Bing-Jie, ; ZHOU Guo-Hua,

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通讯作者: 陈斌, bchen63@163.com

中国化学会秘书处

核酸侵入反应偶联纳米金探针杂交显色技术可视化检测乙醛脱氢酶2基因多态性位点

通讯作者: 邹秉杰,E-mail:zbj523@163.com; 周国华,E-mail:ghzhou@nju.edu.cn

English

Visualized Detection of Aldehyde Dehydrogenase 2 Gene Polymorphism by Serial Invasive Reaction Coupled with Gold Nanoparticle Probe Assembling

Corresponding author: ZOU Bing-Jie, ; ZHOU Guo-Hua,

CCS Chemistry：嵌段共聚物自组装成 “三明治”二维有机材料，实现纳米级压力传感功能

CCS Chemistry：颜徐州、鲍哲南： 你的皮肤可能是一片SPMs

CCS Chemistry：工作高效不疲劳，只须让催化剂动起来

CCS Chemistry：静电组装导向聚合- 聚电解质纳米凝胶量化制备新策略

CCS Chemistry：金黄色葡萄球菌醛基脱氢酶是如何识别特异性底物的？

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