Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-carediagnosis
Isothermal strand displacement amplification (iSDA): a rapid and sensitive method of nucleic acid amplification for point-of-carediagnosis
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DOI：
10.1039/c5an01632k
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发表时间：
2015-01-01
期刊：
影响因子：
4.2
通讯作者：
Yagera, Paul
中科院分区：
文献类型：
--
作者：
Toley, Bhushan J.;Covelli, Isabela;Yagera, Paul
我们提出了一种无需模板初始热变性的DNA快速等温扩增方法，以及用于(a)实时荧光检测和(b)扩增子横向流动检测的方法和探针。等温链位移扩增（iSDA）在49℃条件下，可在20分钟内实现目标序列的bb0 ~ 109倍扩增，是现有最快的等温DNA扩增方法之一。iSDA启动于DNA碱基对自发打开或瞬间转化为胡格斯汀对的位点。“呼吸”，并通过单链的重复划痕、延伸和位移进行指数级放大。我们展示了成功的iSDA扩增和10个金黄色葡萄球菌基因NO的侧流检测。-诱导的l -乳酸脱氢酶（ldh1） （Richardson, Libby, and Fang, Science, 2008, 319, 1672-1676），在清洁样品中，在高浓度基因组DNA和粘蛋白存在下，在< 30分钟内复制50份。我们还提出了一个简单的iSDA动力学模型，其中包含了目标和引物二聚体扩增之间的竞争。这是第一个量化在等温扩增反应中引物-二聚体产物影响的模型。最后，我们通过同时扩增目标基因和工程内控序列来证明iSDA的多路复用能力。iSDA的速度、灵敏度和特异性使其成为即时分子诊断的有力方法。
We present a method of rapid isothermal amplification of DNA without initial heat denaturation of the template, and methods and probes for (a) real-time fluorescence detection and (b) lateral flow detection of amplicons. Isothermal strand displacement amplification (iSDA) can achieve > 109-fold amplification of the target sequence in < 20 minutes at 49 degrees C, which makes it one of the fastest existing isothermal DNA amplification methods. iSDA initiates at sites where DNA base pairs spontaneously open or transiently convert into Hoogsteen pairs, i.e. "breathe", and proceeds to exponential amplification by repeated nicking, extension, and displacement of single strands. We demonstrate successful iSDA amplification and lateral flow detection of 10 copies of a Staphylococcus aureus gene, NO.-inducible L-lactate dehydrogenase (ldh1) (Richardson, Libby, and Fang, Science, 2008, 319, 1672-1676), in a clean sample and 50 copies in the presence of high concentrations of genomic DNA and mucins in < 30 minutes. We also present a simple kinetic model of iSDA that incorporates competition between target and primer-dimer amplification. This is the first model that quantitates the effects of primer-dimer products in isothermal amplification reactions. Finally, we demonstrate the multiplexing capability of iSDA by the simultaneous amplification of the target gene and an engineered internal control sequence. The speed, sensitivity, and specificity of iSDA make it a powerful method for point-of-care molecular diagnosis.