基于CRISPR/Cas12b的乳粉中克罗诺杆菌属检测  

Detection of Cronobacter spp in Milk Powder by CRISPR/Cas12b

作　　者：张懿翔 张清平[1] 李林显 王亮 杨捷琳 刘洋[1] Zhang Yixiang;Zhang Qingping;Li Linxian;Wang Liang;Yang Jielin;Liu Yang(Shanghai Institute of Quality Inspection and Technical Research/Key Laboratory of Milk and Dairy Products Detection and Monitoring Technology for State Market Regulation,Shanghai 200233;Tolo Biotech,Shanghai 200233;Technical Center for Animal,Plant and Food Inspection and Quarantine,Shanghai Customs,Shanghai 200135)

机构地区：[1]上海市质量监督检验技术研究院/国家市场监管重点实验室(乳及乳制品检测与监控技术),上海200233 [2]吐露港生物科技有限公司,上海200233 [3]上海海关动植物与食品检验检疫技术中心,上海200135

出　　处：《中国食品学报》2022年第6期267-275,共9页Journal of Chinese Institute Of Food Science and Technology

基　　金：国家市场监督管理总局技术保障专项项目(2019YJ018)。

摘　　要：目的:开发一种基于CRISPR/Cas12b的克罗诺杆菌属分子检测方法。方法:以克罗诺杆菌菌属特异性基因序列为候选区域,针对区域设计扩增引物和向导RNA靶向序列,对其进行条件优化、特异性检测、反应体系优化,并用人工污染样品对体系的灵敏度、重复性进行验证。结果:该方法的特异性很好,方法的灵敏度达0.01 ng/μL,在人工污染样品中可检测到初始菌量<10 CFU/g的样品,方法检出限<10 CFU/100 g。对市售的51份乳粉样品进行检测,结果与传统国标方法一致。结论:建立的CRISPR/Cas12b方法可用于乳粉中克罗诺杆菌属的快速检测。Objective:The development of a method for detecting Cronobacter spp in milk powder based on CRISPR/Cas12b.Methods:Firstly,the amplification primers and guide RNAs were designed and synthesized based on the specific gene sequence of Cronobacter spp.The conditions,specificity and reaction system were optimized,and the sensitivity and reproducibility of the system were validated using artificially contaminated samples.Results:The results showed a high specificity and sensitivity of this method-the sensitivity reached 0.01 ng/μL;samples with initial bacterial counts less than 10 CFU/g could be detected;and the detection limit was<10 CFU/100 g.51 commercial milk powder samples were later tested using our established method,and the results were consistent with those obtained by traditional national standard method.Conclusion:As such,the CRISPR/Cas12b method established in this study can be used for rapid detection of Cronobacter in milk powder.

关 键 词：CRISPR Cas12b 克罗诺杆菌属 检测 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]