摘要
为实现食品中沙门氏菌的快速检测,选用文献报道的沙门氏菌RPA引物和探针,建立酶促等温扩增(ERA)荧光法和显色法两种沙门氏菌可视化快速检测方法,并分析两种方法的特异性、灵敏度、检出限等指标。同时,为满足实验室外现场检测环境的要求,分别采用自热包和热帖作为现场DNA提取和ERA反应的热源,建立不依赖实验室设备的方法。优化后两种ERA方法检测时间分别为11 min和4 min,灵敏度均可达10^(-2) ng/μL,人工污染样品增菌培养4 h后检出限为1 CFU/mL。本研究创新性地将沙门氏菌RPA引物探针应用到ERA检测体系,建立了荧光法和显色法两种可视化快速检测方法,并集成现场DNA提取和ERA反应检测套组,提高了扩增效率,摆脱了对传统实验室设备的依赖,对于推进食源性致病菌现场可视化快速检测具有重要意义。
In order to realize the rapid detection of Salmonella in food,in this study,the Salmonella RPA primers and probes reported in the literature were used to establish the enzymatic recombinase amplification(ERA)fluorescence method and chromogenic method for visualization of Salmonella.Then,the specificity,sensitivity and detection limit of the two methods were analyzed.Furthermore,considering the requirements of the on-site detection environment outside the laboratory,the self-heating box and heat stickers were used as heat sources for DNA extraction and ERA reaction on-site,respectively.The optimized detection time was only 11 min and 4 min respectively,and the sensitivity was up to 10^(-2) ng/μL.The detection limit of artificially contaminated samples was 1 CFU/mL after culture for 4 h.This study innovatively applied RPA primers and probe applied to ERA detection system,established two visual rapid detection methods:fluorescence method and chromogenic method,and integrated on-site DNA extraction and ERA reaction devices.In this study,the Salmonella RPA primer probe was innovatively applied to the ERA detection system,two visual and rapid detection methods,fluorescence method and chromogenic method were established,and the on-site DNA extraction and ERA reaction detection kit were integrated to improve the amplification efficiency.And get rid of the dependence on traditional laboratory equipment,it is of great significance to promote the rapid detection of food-borne pathogens on-site visualization.
作者
杨艳歌
王帅
李红娜
李涛
吴占文
孙冬梅
袁飞
Yang Yange;Wang Shuai;Li Hongna;Li Tao;Wu Zhanwen;Sun Dongmei;Yuan Fe(Key Laboratory of Food Quality and Safety for State Market Regulation,Chinese Academy of Inspection and Quarantine,Beijing 100176;College of Life Science and Technology,Heilongjiang Bayi Agricultural University,Daqing 163000,Heilongjiang)
出处
《中国食品学报》
EI
CAS
CSCD
北大核心
2023年第10期261-272,共12页
Journal of Chinese Institute Of Food Science and Technology
基金
国家重点研发计划项目(2022YFF0607900,2018YFC1603606)。
