基于微流控芯片的等温扩增技术

Isothermal amplification technology based on microfluidic chip

摘要:

基于聚合酶链式反应(polymerase chain reaction,PCR)的核酸扩增技术是分子诊断领域的金标准，然而PCR往往包含多个反应温度，涉及长时间的循环升降温过程，且需要在复杂热循环仪中完成，这些都限制了其在现场即时检测(point-of-care testing,POCT)中的应用。与传统PCR相比，等温扩增依靠恒定反应温度，反应时间短，检测装置简单，能够提供更加方便、快捷的核酸检测。基于微流控技术的等温扩增检测，通过兼顾微流控与等温扩增两者的优势，能够为POCT分子诊断提供更具竞争力的平台。例如，在新型冠状病毒肺炎(COVID-19)疫情防控中，多种形式的POCT等温扩增检测展示了其独特优势。文中首先归纳总结了典型的等温扩增技术及其检测方法，然后对不同类型的等温扩增微流控系统进行了分类总结与分析(如功能定位、结构组成、流体控制、系统特点等)，最后总结了等温扩增微流控系统在新冠病毒(SARS-CoV-2)等不同病原体检测领域中的应用，并对等温扩增与CRISPR基因编辑等其他新型技术的相互结合进行了介绍与展望。

Abstract:

Polymerase chain reaction (PCR) is the gold standard for nucleic acid amplification in molecular diagnostics.The PCR includes multiple reaction stages (denaturation,annealing,and extension),and a complicated thermalcycler is required to repetitively provide different temperatures for different stages for 30–40 cycles within at least 1–2 hours.Due to the complicated devices and the long amplification time,it is difficult to adopt conventional PCR in point-of-care testing (POCT).Comparing to conventional PCR,isothermal amplification is able to provide a much faster and more convenient nucleic acid detection because of highly efficient amplification at a constant reaction temperature provided by a simple heating device.When isothermal amplification is combined with microfluidics,a more competent platform for POCT can be established.For example,various diagnosis devices based on isothermal amplification have been used to rapidly and conveniently detect SARS-CoV-2 viruses.This review summarized the recent development and applications of the microfluidics-based isothermal amplification.First,different typical isothermal amplification methods and related detection methods have been introduced.Subsequently,different types of microfluidic systems with isothermal amplification were discussed based on their characteristics,for example,functionality,system structure,flow control,and operation principles.Furthermore,detection of pathogens (e.g.SARS-CoV-2 viruses) based on isothermal amplification was introduced.Finally,the combination of isothermal amplification with other new technologies,e.g.CRISPR,has been introduced as well.