等温扩增荧光技术用于检测阪崎肠杆菌的研究  

Study on the Use of Isothermal Amplification Fluorescence Technology for the Detection of Enterobacter sakazakii

作　　者：段真文 许绍坤 姜仁普 梁振瑞 张薇 DUAN Zhenwen;XU Shaokun;JIANG Renpu;LIANG Zhenrui;ZHANG Wei(Yunnan Rare Biotechnology Co.,Ltd.,Kunming 650000,China)

机构地区：[1]云南瑞栢泰生物科技有限公司,云南昆明650000

出　　处：《现代食品》2025年第5期161-164,共4页Modern Food

基　　金：2021年云南省科技厅科技计划项目“分子检测诊断产品关键技术研发”(202102AA310028)。

摘　　要：目的:针对阪崎肠杆菌建立一种快速简便的检测方法。方法:通过对阪崎肠杆菌Esa16S基因序列保守区设计引物,建立阪崎肠杆菌的检测方法;采用直扩法提取核酸,通过引物筛选、最佳反应体系、反应条件的优化、特异性验证与常规普通聚合酶链式反应(Polymerase Chain Reaction,PCR)进行灵敏度对比实验。结果:EBSZ-2为最佳引物组;最佳反应体系配比为15∶6∶4;最佳反应温度为65℃。与常规PCR检测法比较,等温扩增荧光技术的灵敏度为5.76×10^(-4)ng·μL^(-1),是常规方法的1000倍且具有良好的特异性。结论:等温扩增荧光技术可以用来检测阪崎肠杆菌,该技术具有特异性强、灵敏度高、操作简单等优点,能实现快速检测和现场检测。Objective:To establish a rapid and simple detection method for Enterobacter sakazakii.Method:The detection method of Enterobacter sakazakii was established by designing primers for the conserved region of Esa16S gene sequence of Enterobacter sakazakii;nucleic acid was extracted by direct amplification,and the sensitivity was compared with that of conventional polymerase chain reaction(PCR)by primer screening,optimal reaction system,optimization of reaction conditions,specificity verification.Result:The results showed that EBSZ-2 was the best primer set;the optimal reaction system ratio was 15∶6∶4;the optimal reaction temperature was 65℃.Compared with the conventional PCR detection method,the sensitivity of isothermal amplification fluorescence technology was 5.76×10^(-4)ng·μL^(-1),which was 1000 times higher than the conventional method and had good specificity.Conclusion:isothermal amplification fluorescence technology can be used to detect Enterobacter sakazakii,which has the advantages of strong specificity,high sensitivity,simple operation,and can realize rapid detection and on-site detection.

关 键 词：∶等温扩增荧光技术 阪崎肠杆菌 特异性 灵敏度 

分 类 号：TS207.4[轻工技术与工程—食品科学]