Propidium monoazide (PMA) or ethidium bromide monoazide (EMA) treatment has been used before nucleic acid detection methods, such as PCR, to distinguish between live and dead cells using membrane integrity as viability criterion. The performance of these DNA intercalating dyes was compared in many studies utilizing different microorganisms. These studies demonstrated that EMA and PMA differ in their abilities to identify nonviable cells from mixed cell populations, depending on the microorganism and the nature of the sample. Due to this heterogeneity, both dyes were used in the present study to specifically distinguish dead from live Candida albicans cells using viable quantitative PCR (qPCR). The viable qPCR was optimized, and the best results were obtained when pre-treating the cells for 10 min in the dark with 25 μM EMA followed by continuous photoactivation for 15 min. The suitability of this technique to distinguish clotrimazole- and fluconazole-treated C. albicans cells from untreated cells was then assessed. Furthermore, the antifungal properties of two commercial essential oils (Thymus vulgaris and Matricaria chamomilla) were evaluated. The viable qPCR method was determined to be a feasible technique for assessing the viability of C. albicans after drug treatment and may help to provide a rapid diagnostic and susceptibility testing method for fungal infections, especially for patients treated with antifungal therapies.

译文

在核酸检测方法 (例如PCR) 之前,已使用单叠氮化丙啶 (PMA) 或溴化乙锭单叠氮化物 (EMA) 处理,以膜完整性作为生存标准来区分活细胞和死细胞。在许多利用不同微生物的研究中比较了这些DNA嵌入染料的性能。这些研究表明,根据微生物和样品的性质,EMA和PMA在从混合细胞群体中识别非存活细胞的能力上有所不同。由于这种异质性,在本研究中使用了两种染料,以使用可行的定量PCR (qPCR) 特异性区分死亡的白色念珠菌细胞。优化了可行的qPCR,并在黑暗中用25μm EMA预处理细胞10分钟,然后连续光活化15分钟,可获得最佳结果。然后评估了该技术区分克霉唑和氟康唑处理的白色念珠菌细胞与未处理细胞的适用性。此外,还评估了两种商业精油 (百里香和洋甘菊) 的抗真菌特性。可行的qPCR方法被确定为评估药物治疗后白色念珠菌生存能力的可行技术,并可能有助于提供一种快速诊断和药敏试验方法,特别是对于接受抗真菌治疗的患者。

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