SD-PMA-ddPCR检测食品中单核细胞增生李斯特氏菌

Detection of Listeria monocytogenes cells in food based on SD-PMA-ddPCR

摘要:

【目的】检测食品中单核细胞增生李斯特氏菌活菌。【方法】利用脱氧胆酸钠(SD)对受损细胞预处理，然后使叠氮溴化丙锭(PMA)进入受损细胞与DNA发生共价交联，提取细菌基因组DNA进行微滴式数字PCR (ddPCR)检测。【结果】0.1% SD和5.0 mg/L PMA协同作用，可以有效抑制108 CFU/mL的单核细胞增生李斯特氏菌死菌DNA的PCR扩增。经过SD和PMA对样品预处理，ddPCR可以在死菌存在条件下，定量检测鸡肉中单核细胞增生李斯特氏菌活菌，消除了“假阳性”结果的出现。活菌灵敏度检测结果显示：SD-PMA-ddPCR的灵敏度为2.0 copies/20 μL。SD-PMA-ddPCR方法精密度和稳定性良好。【结论】SD-PMA-ddPCR在检测食源性致病菌方面有巨大的发展空间。

Abstract:

[Objective] A method to detect live Listeria monocytogenes cells was researched in the paper. [Methods] Propidium monoazide (PMA) permeated in the injured cells which were treated by sodium deoxycholate (SD), then PMA and DNA conducted covalent cross-linking reaction. Finally, bacterial genome DNA was extracted to detect by ddPCR. [Results] 0.1% SD and 5.0 mg/L PMA could inhibit the DNA PCR amplification of 108 CFU/mL dead L. monocytogenes cells. Only live L. monocytogenes cells were detected by SD-PMA-ddPCR even in the existence of dead L. monocytogenes cells in the chicken. The detection limit is 2.0 copies/20 μL. SD-PMA-ddPCR showed better accuracy and stability. [Conclusion] SD-PMA-ddPCR showed huge potential in foodborne pathogenic bacteria detection.