基于CRISPR-Cas13的单增李斯特菌RNA快速检测方法的建立

Establishment of A Rapid RNA Detection Method for Listeria monocytogenes Based on CRISPR-Cas13

摘要:

为实现食品中单增李斯特菌污染的快速检测，本研究构建了一种基于CRISPR-Cas系统和Broccoli适配体的RNA均相检测技术。利用Cas 13与crRNA锚定序列结合形成识别元件crRNA-Cas13复合物，靶标RNA存在时可激活Cas 13的非特异性RNase活性，并利用点亮型RNA适配体Broccoli作为信号探针，监测crRNA此-Cas13的活化状态。荧光值的变化与单增李斯特菌浓度存在线性关系,利用来检测单增李斯特菌。本研究所构建的检测可在30 min内完成对于单增李斯特菌的的识别与检测，检出限为148 CFU/mL，对细菌具有良好的检测特异性，可区分大肠杆菌、鼠伤寒沙门氏菌和蜡样芽孢杆菌。在牛奶模型中单增李斯特菌的加标回收率为95.15%~97.99%。该方法具有较好的灵敏度、特异性，可直接靶向检测致病菌RNA，无需逆转录、PCR扩增和核酸标记，简化了实验流程，对于实现食品中单增李斯特菌的现场检测及生物安全控制具有重要意义。

Abstract:

In order to realize the rapid detection of Listeria monocytogenes contaminated in food, a homogeneous RNA detection technology based on the CRISPR-Cas system and Broccoli aptamer was constructed in this study. Cas 13 was combined with the crRNA anchor sequence to form crRNA-Cas13 complex as a recognition element. The presence of target RNA activated the non-specific RNase activity of Cas13, and the light-up RNA aptamer Broccoli was used as the signal probe to monitor the activation state of crRNA-Cas13. There is a linear relationship between the change of fluorescence value and the concentration of Listeria monocytogenes, which is used to detect Listeria monocytogenes. The process for identification and detection of Listeria monocytogenes can be completed within 30 minutes with a detection limit of 148 CFU/mL. This method has good detection specificity for bacteria and can distinguish E. coli, S. enterica, Salmonella typhimurium and B. cereus. The recovery rate was 95.15%~97.99% in the milk model samples spiked with Listeria monocytogenes. Therefore, this method has good sensitivity and specificity, and can directly target the pathogen RNAs without reverse transcription, PCR amplification and nucleic acid labeling, which simplifies the experimental process and is of great of significance for realizing on-site detection and biosafety control of Listeria monocytogenes in food.