Research Center For Nanosensor Molecular Diagnostic & Treatment Technology, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, Guangdong, P. R. China.
Graphene Composite Research Center, College of Chemistry and Environmental Engineering, Shenzhen University, Shenzhen 518060, Guangdong, P. R. China.
Anal Chem. 2022 Jul 12;94(27):9724-9731. doi: 10.1021/acs.analchem.2c01193. Epub 2022 Jun 28.
As a golden partner of recombinase polymerase amplification (RPA), CRISPR/Cas12a has been proven to solve the false-positive problem caused by nonspecific amplification perfectly; meanwhile, its -cleave activity has further enhanced the sensitivity. However, the solution transfer operation after tube cap opening greatly increases the risk of aerosol contamination of amplicon, which is inconsistent with point-of-care (POC) diagnostics requirements. This study proposes a photoactivated CRISPR/Cas12a strategy to achieve one-pot high-sensitivity nucleic acid detection. Using photocleavable complementary ssDNA to block crRNA, RPA amplification can smoothly pass through the exponential interval without being affected by activated Cas12a in the critical early stage. After enough amplicons were produced, the Cas12a test was activated by short bursts of ultraviolet radiation at 365 nm. This one-pot method achieved a sensitivity of 2.5 copies within 40 min. This simple and sensitive one-pot method can effectively avoid amplicon contamination and lower the threshold for molecular diagnostics in POC.
作为重组酶聚合酶扩增（RPA）的黄金搭档，CRISPR/Cas12a 已被证明能完美解决非特异性扩增引起的假阳性问题；同时，其切割活性进一步提高了检测的灵敏度。然而，管盖打开后的移液操作大大增加了扩增子气溶胶污染的风险，这与即时检测（POC）诊断的要求不符。本研究提出了一种光激活的 CRISPR/Cas12a 策略，以实现一次性高灵敏度核酸检测。通过使用光可裂解的互补 ssDNA 来阻断 crRNA，在关键的早期阶段，RPA 扩增可以不受激活的 Cas12a 的影响顺利通过指数期。产生足够的扩增子后，用 365nm 的短紫外线脉冲激活 Cas12a 检测。这种一步法在 40 分钟内达到了 2.5 拷贝的灵敏度。这种简单灵敏的一步法可以有效避免扩增子污染，降低 POC 分子诊断的门槛。
