School of Life Sciences, Shanghai University, Shanghai 200444, China.
Institute of Translational Medicine, Shanghai University, Shanghai 200444, China.
Int J Mol Sci. 2024 Mar 20;25(6):3477. doi: 10.3390/ijms25063477.
() is a food-borne pathogenic bacteria that frequently contaminates animal-derived food and low-temperature preserved food. caused by its infection has a high mortality rate and poses a serious threat to human health. Therefore, it is crucial to establish a sensitive, rapid and easy-to-operate technique. In this study, a Recombinase Aided Amplification (RAA) assisted CRISPR/Cas12a (RAA-CRISPR/Cas12a) fluorescence platform was established for highly sensitive nucleic acid detection of . The established RAA-CRISPR/Cas12a showed high sensitivity and high specificity, with the sensitivity of 350 CFU/mL and 5.4 × 10 ng/μL for pure bacterial solution and genomic DNA, and good specificity for 5 strains of spp. and 14 strains of other common pathogenic bacteria. could be detected at an initial concentration of 2.3 CFU/25g within 2 h of enriching the beef in the food matrix, and this method could be applied to food samples that were easily contaminated with The results of RAA-CRISPR/Cas12a could be observed in 5 min, while the amplification was completed in 20-30 min. The speed and sensitivity of RAA-CRISPR/Cas12a were significantly higher than that of the national standard method. In conclusion, the RAA-CRISPR/Cas12a system established in this study has new application potential in the diagnosis of food-borne pathogens.
空肠弯曲菌是一种食源性致病菌,常污染动物源性食品和低温保存食品。由其感染引起的疾病死亡率较高,对人类健康构成严重威胁。因此,建立一种敏感、快速、易于操作的技术至关重要。本研究建立了一种基于重组酶辅助扩增(RAA)的 CRISPR/Cas12a(RAA-CRISPR/Cas12a)荧光平台,用于空肠弯曲菌核酸的高灵敏度检测。建立的 RAA-CRISPR/Cas12a 具有高灵敏度和高特异性,对纯细菌溶液和基因组 DNA 的灵敏度分别为 350 CFU/mL 和 5.4×10ng/μL,对 5 株空肠弯曲菌和 14 株其他常见致病菌具有良好的特异性。在食品基质中富集牛肉 2 小时内,可检测到初始浓度为 2.3 CFU/25g 的空肠弯曲菌,该方法可应用于易受空肠弯曲菌污染的食品样品。RAA-CRISPR/Cas12a 的结果可在 5 分钟内观察到,而扩增可在 20-30 分钟内完成。RAA-CRISPR/Cas12a 的速度和灵敏度明显高于国家标准方法。总之,本研究建立的 RAA-CRISPR/Cas12a 系统在食源性病原体诊断中有新的应用潜力。