Li Fan, Ye Qinghua, Chen Moutong, Xiang Xinran, Zhang Jumei, Pang Rui, Xue Liang, Wang Juan, Gu Qihui, Lei Tao, Wei Xianhu, Ding Yu, Wu Qingping
Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China; School of Biology and Biological Engineering, South China University of Technology, Guangzhou, China.
Guangdong Provincial Key Laboratory of Microbial Safety and Health, State Key Laboratory of Applied Microbiology Southern China, Guangdong Institute of Microbiology, Guangdong Academy of Sciences, Guangzhou, China.
Anal Chim Acta. 2021 Mar 22;1151:338248. doi: 10.1016/j.aca.2021.338248. Epub 2021 Jan 24.
The CRISPR/Cas12a system has displayed remarkable potential in the development of new methods for nucleic acid detection owing to the trans-cleavage activity of Cas12a. Despite the tremendous development in recent years, existing CRISPR/Cas12a-based methods have several limitations such as the time-consuming process, which takes up to 2 h, and the risk of aerosol contamination during DNA amplicon transfer. Herein, we propose a CRISPR/Cas12a-based fluorescence detection platform named "Cas12aFDet" for rapid nucleic acid detection that overcomes these limitations. By integrating PCR or recombinase-aided amplification (RAA) methods with Cas12a-mediated cleavage in a sealed reaction tube, Cas12aFDet-based detection of amplified products could be accomplished within 15 min, while avoiding amplicon contamination. The detection limits of PCR-based Cas12aFDet and RAA-based Cas12aFDet were determined to be 3.37 × 10 cfu/mL and 1.35 × 10 cfu/mL of Listeria monocytogenes serotype 4c in pure culture, respectively. Most importantly, RAA-based Cas12aFDet exhibited 0.64 aM sensitivity for DNA detection, and showed high specificity for detection of other serotypes of Listeria and non-Listeria strains. Furthermore, the feasibility of the RAA-based Cas12aFDet method was evaluated in spiked and natural samples, enabling the quantitative detection of 1.35 × 10-1.35 × 10 cfu/g fresh grass carp of the target L. monocytogenes serotype 4c, and the results obtained for 22 natural aquatic samples were highly consistent with those of the culture-based serotyping method. The established Cas12aFDet platform is expected to provide a new paradigm for the sensitive and specific detection of pathogens in food safety and clinical diagnosis.
由于Cas12a的反式切割活性，CRISPR/Cas12a系统在开发新型核酸检测方法方面展现出显著潜力。尽管近年来取得了巨大进展，但现有的基于CRISPR/Cas12a的方法存在一些局限性，例如耗时长达2小时的过程，以及在DNA扩增子转移过程中存在气溶胶污染的风险。在此，我们提出了一种基于CRISPR/Cas12a的荧光检测平台“Cas12aFDet”，用于快速核酸检测，该平台克服了这些局限性。通过在密封反应管中将PCR或重组酶辅助扩增（RAA）方法与Cas12a介导的切割相结合，基于Cas12aFDet的扩增产物检测可在15分钟内完成，同时避免扩增子污染。基于PCR的Cas12aFDet和基于RAA的Cas12aFDet对纯培养的4c型单核细胞增生李斯特菌的检测限分别确定为3.37×10 cfu/mL和1.35×10 cfu/mL。最重要的是，基于RAA的Cas12aFDet对DNA检测表现出0.64 aM的灵敏度，并且对其他李斯特菌血清型和非李斯特菌菌株的检测具有高特异性。此外，评估了基于RAA的Cas12aFDet方法在加标样品和天然样品中的可行性，能够定量检测目标4c型单核细胞增生李斯特菌在新鲜草鱼中的含量为1.35×10 - 1.35×10 cfu/g，并且22个天然水产样品的检测结果与基于培养的血清分型方法高度一致。所建立的Cas12aFDet平台有望为食品安全和临床诊断中病原体的灵敏特异检测提供新的范例。
