Visual detection of gene mutations based on isothermal strand-displacement polymerase reaction and lateral flow strip.
Visual detection of gene mutations based on isothermal strand-displacement polymerase reaction and lateral flow strip.
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DOI：
10.1016/j.bios.2011.10.037
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发表时间：
2012-01-15
影响因子：
12.6
通讯作者：
Liu, Guodong
中科院分区：
文献类型：
--
作者：
He, Yuqing;Zeng, Kang;Zhang, Sanquan;Gurung, Anant S.;Baloda, Meenu;Zhang, Xibao;Liu, Guodong
在这里，我们描述了一种简单而灵敏的基于等温链置换聚合酶反应(ISDPR)和横向流条(LFS)的基因突变视觉检测方法。这一概念最初是通过检测表皮松解性角化过度症(EHK)中角蛋白10的R156H突变基因而得到证实的。在生物素修饰的发夹DNA和地高辛修饰的引物存在下，R156H突变DNA触发ISDPR产生大量地高辛和生物素连接的双链DNA产物。在LFS上通过双重免疫反应(金纳米颗粒上的抗地高辛抗体(Au-NP)和双链上的地高辛抗体，LFS试验区上的抗生物素抗体和双链上的生物素)来检测产物。Au-NPs的积累产生了特有的红带，使得无需仪器即可直观检测突变基因。在对ISDPR的实验条件和检测参数进行系统优化后，本方法无需仪器即可在75分钟内检测到低至1-fM的R156H-突变DNA。在ISDPR过程中，通过使用荧光素和生物素修饰的发夹探针，实现了R156突变位点上R156H和R156C突变DNA的区分。因此，该方法为基因突变的检测提供了一种简单、灵敏和低成本的工具。
Here, we describe a simple and sensitive approach for visual detection of gene mutations based on isothermal strand-displacement polymerase reactions (ISDPR) and lateral flow strip (LFS). The concept was first demonstrated by detecting the R156H-mutant gene of keratin 10 in Epidermolytic hyperkeratosis (EHK). In the presence of biotin-modified hairpin DNA and digoxin-modified primer, the R156H-mutant DNA triggered the ISDPR to produce numerous digoxin- and biotin-attached duplex DNA products. The product was detected on the LFS through dual immunoreactions (anti-digoxin antibody on the gold nanoparticle (Au-NP) and digoxin on the duplex, anti-biotin antibody on the LFS test zone and biotin on the duplex). The accumulation of Au-NPs produces the characteristic red band, enabling visual detection of the mutant gene without instrumentation. After systematic optimization of the ISDPR experimental conditions and the parameters of the assay, the current approach is capable of detecting as low as 1-fM R156H-mutant DNA within 75 min without instrumentation. Differentiation of R156H- and R156C-mutant DNA on the R156 mutation site was realized by using fluorescein- and biotin-modified hairpin probes in the ISDPR process. The approach thus provides a simple, sensitive, and low-cost tool for the detection of gene mutations.
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