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【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(dd PCR)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/m L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,dd PCR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-dd PCR的灵敏度为2.0 copies/20μL。SD-PMA-dd PCR方法精密度和稳定性良好。【结论】SD-PMA-dd PCR在检测食源性致病菌方面有巨大的发展空间。
【Objective】 Detection of viable Listeria monocytogenes in food. 【Method】 The damaged cells were pretreated with sodium deoxycholate (SD) and then covalently crosslinked with azido-bromide-propidium (PMA) into damaged cells and DNA. The bacterial genomic DNA was extracted for microdrops numbers PCR (dd PCR) detection. 【Result】 The results showed that the synergistic effect of 0.1% SD and 5.0 mg / L PMA could effectively inhibit the PCR amplification of 108 CFU / mL Listeria monocytogenes DNA. After pretreatment of SD and PMA samples, dd PCR quantifies viable Listeria monocytogenes in chicken in the presence of dead bacteria, eliminating the “false positive” result. Viable sensitivity test results showed that the sensitivity of SD-PMA-dd PCR was 2.0 copies / 20μL. SD-PMA-dd PCR method has good precision and stability. 【Conclusion】 SD-PMA-dd PCR has great potential for detecting food-borne pathogens.