建立将叠氮溴化丙锭(PMA)与定量PCR(qPCR)技术结合，用于检测热灭活菌背景条件下的大肠杆菌活菌的方法。结果表明：5min以上的强烈光照可以使PMA与DNA共价交联，同时完全钝化游离的PMA以避免“假阴性”结果；抑制死菌DNA扩增的最低PMA质量浓度为10μg/mL，不抑制活菌DNA扩增的最高PMA质量浓度为20μg/mL。当活菌/总菌大于1%时，经PMA预处理，可以消除热灭活菌DNA的干扰，实现对活菌的定量检测。%In this work, a method to detect live E. coli O157:H7 was established using propidium monoazide (PMA) coupled with quantitative polymerase chain reaction (qPCR). The minimum inhibitory concentration against DNA amplification from dead bacteria was 10 µg/mL, and PMA did not inhibit DNA application from live bacteria at concentrations equal to or lower than 20 µg/mL. Covalent cross-linking of PMA with DNA was induced by strong light illumination for 5 min and meanwhile, free PMA was completely inactivated, resulting in the avoidance of false negative results. When the live to dead bacteria ratio was more than 1%, PMA pretreatment allowed the elimination of DNA interference from thermally inactivated bacteria and therefore live bacteria could be quantiifed.
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