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Self-Priming Hairpin-Utilized Isothermal Amplification Enabling Ultrasensitive Nucleic Acid Detection.
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-06-19 , DOI: 10.1021/acs.analchem.0c00511 Ja Yeon Song 1 , Yujin Jung 1 , Seoyoung Lee 1 , Hyun Gyu Park 1
Analytical Chemistry ( IF 6.7 ) Pub Date : 2020-06-19 , DOI: 10.1021/acs.analchem.0c00511 Ja Yeon Song 1 , Yujin Jung 1 , Seoyoung Lee 1 , Hyun Gyu Park 1
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We presented a novel method, referred to as self-priming hairpin-utilized isothermal amplification (SPHIA), to enable nucleic acid detection. The hairpin probe (HP1) employed in this strategy was designed to be opened through binding to the target nucleic acid. Upon opening of HP1, the self-priming domain within the HP1 stem region was exposed and rearranged to serve as a primer. The following extension displaced the bound target nucleic acid, which was then recycled to open another HP1. The extended HP1 was subjected to continuously repeated extension and nicking reaction, and abundant triggers were produced as a result. The triggers entered and initiated phase 2 reaction through binding to HP2, and this produced numerous target mimic strands (Target′). The target′ entered and activated the phase 1 reaction, mimicking the target nucleic acid. As a consequence of these interconnected two amplification reactions initiated by a positive feedback mechanism, a considerable number of final double-stranded DNA products (FPs) were ultimately produced, which could be monitored in real-time through duplex-specific fluorescent signaling. Exploiting this novel design principle, we detected target DNA down to 28.9 aM with outstanding discrimination capability. The practical diagnostic ability of this strategy was also successfully demonstrated for target RNA detection by rationally redesigning the hairpin probes.
中文翻译：
自发发夹利用等温扩增实现超灵敏核酸检测。
我们提出了一种新方法，称为自引发发夹利用等温扩增（SPHIA），以实现核酸检测。该策略中采用的发夹探针（HP1）设计为通过与靶核酸结合而打开。HP1打开后，HP1茎区域内的自启动域暴露并重新排列以用作引物。随后的延伸置换了结合的靶核酸，然后将其再循环以打开另一个HP1。延伸的HP1经受连续重复的延伸和切口反应，结果产生了大量的触发物。触发器通过与HP2结合而进入并启动了第2阶段反应，这产生了许多靶标模拟链（Target'）。靶标进入并激活了阶段1反应，模拟了靶标核酸。由于通过正反馈机制引发的两个相互连接的扩增反应相互连接，最终产生了大量最终的双链DNA产物（FP），可通过双链特异性荧光信号进行实时监测。利用这一新颖的设计原理，我们检测到具有出色识别能力的低至28.9 aM的靶DNA。通过合理地重新设计发夹探针，还成功地证明了该策略的实用诊断能力可用于靶RNA检测。利用这一新颖的设计原理，我们检测到具有出色识别能力的低至28.9 aM的靶DNA。通过合理地重新设计发夹探针，还成功地证明了该策略的实用诊断能力可用于靶RNA检测。利用这一新颖的设计原理，我们检测到具有出色识别能力的低至28.9 aM的靶DNA。通过合理地重新设计发夹探针，还成功地证明了该策略的实用诊断能力可用于靶RNA检测。
更新日期：2020-08-04
中文翻译：
自发发夹利用等温扩增实现超灵敏核酸检测。
我们提出了一种新方法，称为自引发发夹利用等温扩增（SPHIA），以实现核酸检测。该策略中采用的发夹探针（HP1）设计为通过与靶核酸结合而打开。HP1打开后，HP1茎区域内的自启动域暴露并重新排列以用作引物。随后的延伸置换了结合的靶核酸，然后将其再循环以打开另一个HP1。延伸的HP1经受连续重复的延伸和切口反应，结果产生了大量的触发物。触发器通过与HP2结合而进入并启动了第2阶段反应，这产生了许多靶标模拟链（Target'）。靶标进入并激活了阶段1反应，模拟了靶标核酸。由于通过正反馈机制引发的两个相互连接的扩增反应相互连接，最终产生了大量最终的双链DNA产物（FP），可通过双链特异性荧光信号进行实时监测。利用这一新颖的设计原理，我们检测到具有出色识别能力的低至28.9 aM的靶DNA。通过合理地重新设计发夹探针，还成功地证明了该策略的实用诊断能力可用于靶RNA检测。利用这一新颖的设计原理，我们检测到具有出色识别能力的低至28.9 aM的靶DNA。通过合理地重新设计发夹探针，还成功地证明了该策略的实用诊断能力可用于靶RNA检测。利用这一新颖的设计原理，我们检测到具有出色识别能力的低至28.9 aM的靶DNA。通过合理地重新设计发夹探针，还成功地证明了该策略的实用诊断能力可用于靶RNA检测。
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