PMA结合ddPCR检测食品中沙门氏菌

Detection of Salmonella in Food Based on PMA-ddPCR

DOI：10.3969/j.issn.1673-1689.2017.10.009

中文关键词: 叠氮溴化丙锭 微滴式数字PCR 沙门氏菌 活菌

英文关键词: Propidium monoazide,ddPCR,Salmonella,live cells

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中文摘要:

建立一种快速、灵敏的微滴式数字PCR方法，用于检测食品中沙门氏菌活菌。利用叠氮溴化丙锭（PMA）对死菌预处理，PMA可以选择性穿过死菌细胞膜，在光照条件下与死菌DNA发生共价交联反应，进而阻止其DNA进行PCR扩增，提取细菌基因组DNA进行微滴式数字PCR（ddPCR）检测。结果表明，强烈光照15.0 min，可以使PMA与死菌DNA共价交联，同时钝化游离的PMA；PMA终质量浓度为40.0 μg/mL可以有效抑制105 CFU/mL的沙门氏菌死菌DNA的PCR扩增；不抑制活菌DNA扩增的PMA最高浓度是50.0 μg/mL。利用PMA处理死活菌混合液时，PMA-ddPCR可以在死菌存在的条件下，定量检测活菌，降低了“假阳性”结果的出现。灵敏度检测结果显示：PMA-ddPCR灵敏度是0.4 copy/μL。利用PMA-ddPCR检测人工污染的鳕鱼样品，最低可检出102 CFU/mL的沙门氏菌，精密度和稳定性良好。

英文摘要:

The research aimed to establish a fast method to detect of live Salmonella in food with high sensitity. Propidium monoazide（PMA） permeated in the injured cells，then PMA and DNA conducted covalent cross-linking reaction，which could inhibit PCR amplification. Finally，bacterial genome DNA was extracted to detect by ddPCR. The optimization of experimental parameters showed that a final PMA concentration of 40.0 μg/mL and exposure of 15 min could restrain DNA amplification of 105 CFU/mL dead cells ，and the maximum PMA against DNA amplification from live Salmonella cells was 50.0 μg/mL. Under the different live/dead bacteria proportion，only live Salmonella cells was detected by PMA-ddPCR even in the existence of dead Salmonella cells. The detection limit was 8.0 copy/20 μL. PMA-ddPCR could detect 102 CFU/mL Salmonella cells in the codfish polluted by manual work. Furthermore，PMA-ddPCR showed better accuracy and stability. In conclusion，PMA-ddPCR showed huge potential in foodborne pathogenic bacteria detection.

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