Selective PCR detection of viable Enterobacter sakazakii cells utilizing propidium monoazide or ethidium bromide monoazide

Aims: The detection of viable Enterobacter sakazakii cells is important due to the association of this pathogen with outbreaks of life-threatening neonatal infections. The aim of this study was to optimize a PCR-based method for selective detection of only viable Ent. sakazakii cells in the presence of dead cells, utilizing propidium monoazide (PMA) or ethidium bromide monoazide (EMA). Methods and Results: PMA or EMA was added to suspensions of viable and/or dead Ent. sakazakii cells at varying concentrations (10, 50 or 100 μg ml⁻¹) prior to DNA isolation and PCR with Ent. sakazakii-specific primers. At concentrations of 50 and 100 μg ml⁻¹, PMA completely inhibited PCR amplification from dead cells, while causing no significant inhibition of the amplification from viable cells. PMA was also effective in allowing selective PCR detection of only viable cells in mixtures of varying ratios of viable and dead cells. EMA was equally effective in preventing amplification from dead cells, however, it also inhibited DNA amplification from viable cells. Conclusions: This study demonstrated the efficiency of PMA for viable and dead differentiation of Ent. sakazakii, as well as the lack of selectivity of EMA for this purpose. Significance and Impact of the Study: PMA-PCR, in particular, will be useful for monitoring the resistance, survival strategies and stress responses of Ent. sakazakii in foods and the environment.     

副溶血弧菌EMA-PCR检测技术的建立

PCR技术被广泛应用于副溶血弧菌的检测中, 但传统的PCR技术无法区分样品中的死细菌与活细菌, 往往使检测结果出现较高的假阳性。因此, 将叠氮溴乙锭(Ethidium monoazide bromide, EMA)与PCR技术结合, 建立一种快速、准确的副溶血弧菌检测方法。以dnaJ基因为检测副溶血弧菌的靶基因, 分别用副溶血弧菌的纯培养细胞及其基因组DNA作模板进行PCR检测, 灵敏度分别为2.5×104 CFU/mL和6×102 fg/μL。在检测样品前处理过程中加入EMA, 当EMA的浓度小于5 mg/L时, EMA对活菌靶基因的扩增没有明显的抑制; 而终浓度为2 mg/L的EMA, 能有效抑制1×108 CFU/mL副溶血弧菌死菌的扩增。活菌和死菌混合体系的PCR结果表明, EMA-PCR能有效降低副溶血弧菌检测过程中的假阳性。