PMA结合ddPCR检测食品中金黄色葡萄球菌的研究  

Detection of Staphylococcus aureus in Foods by PMA Combined with ddPCR

作　　者：赵丽青[1] 王静 秦燕[2] 贾俊涛[1] 姜英辉[1] 唐静[1] 张健[1] 

机构地区：[1]山东出入境检验检疫局,山东青岛266002 [2]威海出入境检验检疫局,山东威海264200

出　　处：《微生物学杂志》2017年第1期105-109,共5页Journal of Microbiology

基　　金：国家质检总局科研项目(2014IK114,2016IK198)

摘　　要：研究了将叠氮溴化丙锭(PMA)与微滴式数字PCR(ddPCR)技术相结合,用于金黄色葡萄球菌活菌的检测。结果表明,强烈光照15 min,可以使PMA与死菌DNA共价交联,同时钝化游离的PMA;可以有效抑制金黄色葡萄球菌死菌DNAPCR扩增的PMA终浓度为2.0μg/m L;不抑制活菌DNA扩增的PMA最高浓度是5.0μg/m L。在不同死、活菌比例下,PMA-ddPCR可以定量检测活菌,避免了死菌DNA的干扰,本方法的检出限为10 copy/20μL。利用PMA-ddPCR检测人工污染鸡肉样品,最低可检出102cfu/m L的金黄色葡萄球菌。表明PMA-ddPCR方法的灵敏度高。Propidium monoazide（ PMA） combined with droplet digital PCR（ ddPCR） was used for testing live Staphylococcus aureus in this study. The results showed that under strong light radiation for 15 min. made the covalent cross linkage between PMA and dead bacteria DNAto happen and free PMA to be deactivated simultaneously; The final PMA concentration of 2. 0 μg/m L can effectively inhibit DNAPCR amplification of dead S. aureus; The highest PMA concentration that cannot inhibit DNAPCR amplification of live S. aureus was 5. 0 μg/m L. Mixture of dead and live S. aureus in different proportion,PMA-ddPCR can quantitatively detect live bacteria and avoid the interference of dead bacteria DNA. The detection limits of the method was 10 copy/20 μL. Using PMA-ddPCR to detect samples of artificial infected chicken,the minimum of S. aureus can be detected was 102cfu/m L. Suggested that PMA-ddPCR method had high sensitivity.

关 键 词：叠氮溴化丙锭 微滴式数字PCR 金黄色葡萄球菌 

分 类 号：Q93[生物学—微生物学] TS207[轻工技术与工程—食品科学]