School of Life Sciences, South China Normal University, Guangzhou 510631, China.
Guangzhou Key Laboratory of analytical Chemistry for Biomedicine, School of Chemistry, South China Normal University, Guangzhou 510006, China.
Anal Chem. 2022 Jun 14;94(23):8277-8284. doi: 10.1021/acs.analchem.2c00616. Epub 2022 May 30.
CRISPR/Cas12, a highly efficient and specific nucleic acid recognition system, has been broadly employed to detect amplified DNA products. However, most reported methods adopt a two-step detection mode that needs a liquid transfer step, thus complicating the detection procedure and posing a risk of aerosol contamination. A one-pot detection method can obviate these problems, but it suffers from poor detection efficiency due to the loss of amplification templates elicited by CRISPR/Cas12 cleavage. In this study, we discovered that a glycerol additive dramatically promoted the detection efficiency of the one-pot recombinase polymerase amplification (RPA)-CRISPR/Cas12a method. Compared with the glycerol-free version, its sensitivity was nearly 100-fold higher and was close to that of the canonical two-step method. Further investigation displayed that the enhanced detection efficiency was attributed to the phase separation of the RPA and CRISPR/Cas12a system during the initial phase of the RPA reaction caused by the glycerol viscosity. This highly efficient one-pot method has been triumphantly harnessed for the detection of African swine fever virus (ASFV) and SARS-CoV-2, achieving naked-eye readout through a smartphone-equipped device. The currently developed glycerol-enhanced one-pot RPA-CRISPR/Cas12a method can be an advantageous point-of-care nucleic acid detection platform on account of its simplicity, high sensitivity, and universality.
CRISPR/Cas12 是一种高效、特异的核酸识别系统，已广泛应用于扩增 DNA 产物的检测。然而，大多数报道的方法采用两步检测模式，需要进行液体转移步骤，从而使检测过程复杂化，并存在气溶胶污染的风险。一步法检测可以避免这些问题，但由于 CRISPR/Cas12 切割引发的扩增模板丢失，其检测效率较差。在本研究中，我们发现甘油添加剂可显著提高一步法重组酶聚合酶扩增（RPA）-CRISPR/Cas12a 方法的检测效率。与无甘油版本相比，其灵敏度提高了近 100 倍，接近于经典的两步法。进一步研究表明，增强的检测效率归因于甘油粘度引起的 RPA 和 CRISPR/Cas12a 系统在 RPA 反应初始阶段的相分离。这种高效的一步法已成功应用于非洲猪瘟病毒（ASFV）和 SARS-CoV-2 的检测，通过配备智能手机的设备实现了肉眼可读的结果。目前开发的甘油增强型一步法 RPA-CRISPR/Cas12a 方法具有简单、高灵敏度和通用性等优点，有望成为一种有利的即时检测核酸检测平台。
