Cawthorn D-M, Witthuhn R C
Department of Food Science, University of Stellenbosch, Matieland, South Africa.
J Appl Microbiol. 2008 Oct;105(4):1178-85. doi: 10.1111/j.1365-2672.2008.03851.x. Epub 2008 Jul 8.
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).
PMA or EMA was added to suspensions of viable and/or dead Ent. sakazakii cells at varying concentrations (10, 50 or 100 microg ml(-1)) prior to DNA isolation and PCR with Ent. sakazakii-specific primers. At concentrations of 50 and 100 microg 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.
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.
PMA-PCR, in particular, will be useful for monitoring the resistance, survival strategies and stress responses of Ent. sakazakii in foods and the environment.
由于阪崎肠杆菌与危及生命的新生儿感染暴发有关，因此检测其活细胞具有重要意义。本研究的目的是优化一种基于聚合酶链反应（PCR）的方法，利用单叠氮化丙锭（PMA）或单叠氮化溴化乙锭（EMA）在存在死细胞的情况下选择性检测仅存活的阪崎肠杆菌细胞。
在进行DNA提取及使用阪崎肠杆菌特异性引物进行PCR之前，将不同浓度（10、50或100μg/ml）的PMA或EMA添加到存活和/或死亡的阪崎肠杆菌细胞悬液中。在50和100μg/ml的浓度下，PMA完全抑制了死细胞的PCR扩增，同时对活细胞的扩增没有显著抑制作用。PMA在不同活细胞与死细胞比例的混合物中也能有效地实现仅对活细胞的选择性PCR检测。EMA在阻止死细胞扩增方面同样有效，然而，它也抑制了活细胞的DNA扩增。
本研究证明了PMA对阪崎肠杆菌活细胞和死细胞鉴别的有效性，以及EMA在此方面缺乏选择性。
特别是PMA-PCR将有助于监测阪崎肠杆菌在食品和环境中的抗性、生存策略及应激反应。
