Recombinase assisted loop-mediated isothermal DNA amplification
Recombinase assisted loop-mediated isothermal DNA amplification
复制标题
重组酶辅助环介导的等温 DNA 扩增
DOI：
10.1039/c9an01701a
复制
发表时间：
2020-01-21
期刊：
影响因子：
4.2
通讯作者：
Tang, Zhuo
中科院分区：
文献类型：
--
作者：
Chen, Gangyi;Chen, Rong;Tang, Zhuo
聚合酶链反应（PCR）和等温扩增方法如LAMP和RPA广泛用于基因检测。然而，这些方法也存在一些缺点，如对热循环仪的依赖性、引物设计的复杂性、LAMP中非特异性扩增的可能性以及RPA中组分的复杂性。我们建立了一种新的等温DNA检测系统，命名为酶辅助环介导扩增（RALA）。使用来自嗜热栖热菌（Thermus thermophilus）的内切酶（TthRecA）来打开靶双链DNA以在等温条件下启动环介导的扩增，这简化了引物设计并避免了预变性。引入名为ProofMan的FRET传感器和校正酶Pfu以通过从3 '端切割传感器来产生荧光信号。因此，实现了基于RALA系统的序列特异性检测，甚至可以鉴定单核苷酸多态性（SNP）。通过引入额外的环引物，快速RALA版本可以在30分钟内扩增10（2）个DNA靶标。除了高灵敏度和特异性之外，选择不同报告传感器的灵活性使得该方法在定量或定性DNA检测中通用。
Polymerase chain reaction (PCR) and isothermal amplification methods such as LAMP and RPA are widely used for genetic detection. However, there are some shortcomings of these methods such as dependence on thermocycler instruments for PCR, complexity of primer design, the possibility for nonspecific amplification in LAMP and complexity of components in RPA. We develop a novel isothermal DNA detection system named Recombinase Assisted Loop-mediated Amplification (RALA). Recombinase from Thermus thermophilus (TthRecA) was used to open target double-stranded DNA to initiate loop-mediated amplification under isothermal conditions, which simplified the primer design and circumvented pre-denaturation. A FRET sensor named ProofMan and a proofreading enzyme Pfu were introduced to produce fluorescence signals by cleaving the sensor from the 3 ' end. Consequently, sequence-specific detection based on the RALA system was achieved, and even a single nucleotide polymorphism (SNP) could be identified. By introducing additional loop primers, the fast RALA version can amplify 10(2) DNA targets in 30 minutes. In addition to high sensitivity and specificity, the flexibility of choosing different reporting sensors makes this method versatile in either quantitative or qualitative DNA detection.