恒温实时荧光法检测金黄色葡萄球菌  

Detection of Staphyloccocus aureus by Real Time Fluorescence Isothermal Amplicication

作　　者：刘羽霏[1] 叶蕾 孟赫诚[3] 闫鹤[3] 石磊[3] 

机构地区：[1]华南农业大学食品学院,广东广州510642 [2]广州迪澳生物科技有限公司,广东广州510663 [3]华南理工大学轻工与食品学院,广东广州5106403

出　　处：《现代食品科技》2013年第9期2262-2266,共5页Modern Food Science and Technology

基　　金：中央高校基本科研业务费专项资金资助项目(2012ZZ0083);国家自然科学基金青年科学基金项目(31201363);广州市科技计划项目(11C12080718)

摘　　要：恒温实时荧光技术是目前最新颖的核酸扩增方法。本文创建了恒温实时荧光法快速检测微生物技术方法并应用于金黄色葡萄球菌(Staphyloccocus aureus)的快速检测。本研究针对金葡菌特异序列nuc设计6条引物,选取常见病原菌标准株为对照品进行引物特异性检测;选取金葡菌标准菌株进行灵敏度检测;并且利用食品中分离的金葡菌,同时进行LAMP反应和荧光PCR实验,验证LAMP反应的检出率,结合ESEQuant tube scanner进行实时检测。结果显示:所建立的恒温荧光扩增法具有扩增效率高、特异性强、灵敏度高(灵敏度为102CFU/mL)等优点。对50食品中分离的金葡菌进行检测,恒温荧光法检测出50株,荧光PCR的方法检测出50株,两种方法的检出率都为100%。本文引入的ESE Quant tube scanner平台结合恒温实时荧光法检测金葡菌不仅操作简单,便于携带,同时实现检测过程的数据化及自动化,适合金葡菌的现场检测和大规模监控。Isothermal real-time fluorescence technology is one of the most novel nucleic acid amplification method. 6 primers targeting the nuc gene of Staphyloccocus aureus was applied to detect S. aureus with ESEQuant tube scanner; Specificity of the primers was examined using stander strains of S. aureusand non-S, aureusstmins.S, aureus isolated from food was detected by LAMP based assay and by real time PCR. The results showed that the real time fluorescence isothermal amplicication had good efficiency, high specificity and high sensitivity （10z CFU/mL）. 50 Staphyloccocus aureus strains isolated from food samples were selected for stability tests. 50 strains was detected as Staphyloccocus aureuswith LAMP and 50 strains were identified with real time PCR. The results in this study suggested that the ESE tube scanner platform has great potential as a field usable molecular tool for diagnosis of Staphyloccocus aurem＇. In addition, it provide an alternative diagnostic methods for field set, and for detection in developing area.

关 键 词：金黄色葡萄球菌 环介导等温扩增 

分 类 号：R155.5[医药卫生—营养与食品卫生学]