School of Food Science and Technology, International Joint Laboratory on Food Safety, Synergetic Innovation Center of Food Safety and Quality Control, Jiangnan University, Wuxi, Jiangsu, 214122, PR China; Yixing Institute of Food and Biotechnology Co., Ltd, Yixing, 214200, PR China.
School of Food Science and Technology, International Joint Laboratory on Food Safety, Synergetic Innovation Center of Food Safety and Quality Control, Jiangnan University, Wuxi, Jiangsu, 214122, PR China; Yixing Institute of Food and Biotechnology Co., Ltd, Yixing, 214200, PR China.
Anal Chim Acta. 2024 Feb 15;1290:342203. doi: 10.1016/j.aca.2024.342203. Epub 2024 Jan 6.
Foodborne pathogenic bacteria are widespread in various foods, whose cross-contamination and re-contamination are critical influences on food safety. Rapid, accurate, and sensitive detection of foodborne pathogenic bacteria remains a topic of concern. CRISPR/Cas12a can recognize double-stranded DNA directly, showing great potential in nucleic acid detection. However, few studies have investigated the cleavage properties of CRISPR/Cas12a. In this study, the trans-cleavage properties of LbCas12a and AsCas12a were investigated to construct the detection methods for foodborne pathogenic bacteria. The highly sensitive fluorescent strategies for foodborne pathogens were constructed by analyzing the cleavage rates and properties of substrates at different substrate concentrations. Cas12a was activated in the presence of foodborne pathogenic target sequence was present, resulting in the cleavage of a single-stranded reporter ssDNA co-labelled by fluorescein quencher and fluorescein. The sensitivity and specificity of the Cas12a fluorescent strategy was investigated with Salmonella and Staphylococcus aureus as examples. The results showed that AsCas12a was slightly more capable of trans-cleavage than LbCas12a. The detection limits of AsCas12a for Salmonella and Staphylococcus aureus were 24.9 CFU mL and 1.50 CFU mL, respectively. In all the seven bacteria, Staphylococcus aureus and Salmonella were accurately discriminated. The study provided a basis for constructing and improving the CRISPR/Cas12a fluorescence strategies. The AsCas12a-based detection strategy is expected to be a promising method for field detection.
食源性病原体广泛存在于各种食品中，其交叉污染和再污染对食品安全是至关重要的影响因素。快速、准确、灵敏地检测食源性病原体仍然是一个关注的话题。CRISPR/Cas12a 可以直接识别双链 DNA，在核酸检测中显示出巨大的潜力。然而，关于 Cas12a 的切割特性的研究较少。在这项研究中，研究了 LbCas12a 和 AsCas12a 的转切割特性，以构建食源性病原体的检测方法。通过分析不同底物浓度下底物的切割率和特性，构建了食源性病原体的高灵敏度荧光检测策略。当存在食源性病原体靶序列时，Cas12a 被激活，导致荧光猝灭剂和荧光标记的单链报告 ssDNA 被切割。以沙门氏菌和金黄色葡萄球菌为例，研究了 Cas12a 荧光策略的灵敏度和特异性。结果表明，AsCas12a 的转切割能力略优于 LbCas12a。AsCas12a 对沙门氏菌和金黄色葡萄球菌的检测限分别为 24.9 CFU mL 和 1.50 CFU mL。在所有七种细菌中，金黄色葡萄球菌和沙门氏菌均可被准确区分。该研究为构建和改进 Cas12a 荧光策略提供了依据。基于 AsCas12a 的检测策略有望成为现场检测的一种有前途的方法。
