超灵敏高分辨的Digital-CRISPR技术用于免扩增的多重核酸检测

高灵敏、高特异性的核酸检测在临床检测、食品安全、环境监测等方面具有重要意义。传统的分子诊断通常需要借助核酸扩增技术，由此带来的流程繁琐、操作复杂、需集中送检、易导致气溶胶污染等限制无法满足当前检测的需求，尤其是此次COVID-19的爆发使这一问题更加明显。因此，目前亟需开发出免扩增的分子诊断新技术用于核酸分子的超灵敏检测。近些年来兴起的CRISPR/Cas技术具有超高的特异性以及反式切割效率，在此基础上，本项目借助阵列式Digital微流控芯片的信号放大能力以及Multi-crRNA识别技术进一步降低检测限实现核酸分子的超灵敏检测；并且还将充分发挥CRISPR/Cas技术的超高特异性用于多重核酸分子的同时多色检测。本项目提出的免扩增的Digital-CRISPR核酸检测技术可在30 min内实现1 aM核酸分子的快速精准检测，在临床诊断、食品安全、环境监测等领域具有重大的意义及实用价值。

High sensitive and specific nucleic acid detection is of great significance in clinical detection, food safety, environmental monitoring, etc. Traditional molecular diagnostics usually rely on nucleic acid amplification technology, which can not meet current detection requirements due to the complicated process, complex operation, centralized testing, and easily causing aerosol pollution, especially the outbreak of COVID-19 has made this problem more pronounced. Therefore, it is urgent to develop new amplification-free molecular diagnostic techniques for ultrasensitive detection of nucleic acid molecules. The emerging CRISPR/Cas technology in recent years has high specificity and trans-cleavage efficiency. On this basis, this project further reduces the detection limit to achieve the ultra-sensitive detection of nucleic acid molecules with the help of the signal amplification ability of digital microfluidic chip and the multi-crRNA recognition technology. In addition, it will give full play to the ultra-high specificity of CRISPR/Cas technology for simultaneous multicolor detection of multiple nucleic acid molecules. The amplification-free Digital-CRISPR nucleic acid detection technology proposed in this project can achieve rapid and accurate detection of 1 aM nucleic acid molecules within 30 min, which has great significance and practical value in clinical diagnosis, food safety, environmental monitoring, and other fields.