Quantification of a cell culture contaminant using 16S rDNA.
Quantification of a cell culture contaminant using 16S rDNA.
复制标题
使用 16S rDNA 对细胞培养物污染物进行定量。
DOI：
10.1002/bab.1792
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发表时间：
2019
影响因子：
2.8
通讯作者：
Hua Zichun
中科院分区：
文献类型：
--
作者：
Xu Xuebo;Lai Yueyang;Zhou Wenzhao;Wu Leyang;Hua Zichun
在这项研究中，我们使用16 S rDNA测序将一种“黑点”样细胞培养污染物鉴定为属于Pusillimonas属的物种。在所有测试的抗生素中，氨苄青霉素和庆大霉素（均为100 µg/mL）的联合治疗能够消除这种污染物。然后通过使用碘化丙啶染色的荧光显微镜观察污染物，并在污染的A549细胞的胞质溶胶内发现污染物。为了表征抗生素去除污染物的功效，我们设计了一种定量方法来确定与单个A549细胞相关的16 S rDNA拷贝的平均数量，其与每个A549细胞的污染物的平均数量成正比。通过使用对污染物的16 S rDNA序列具有特异性的引物，我们能够使用基于qPCR的相对和绝对定量来估计每个污染细胞的污染物。
In this study, we identified a “black dot”‐like cell culture contaminant as a species belonging to the genus ofPusillimonasusing 16S rDNA sequencing. Among all antibiotics tested, a combinatorial treatment of ampicillin and gentamicin both at 100 µg/mL was able to eliminate this contaminant. The contaminant was then visualized by fluorescence microscopy using propidium iodide staining and was found inside the cytosol of contaminated A549 cells. To characterize the efficacy of antibiotics for contaminant removal, we devised a quantitative method to determine the average number of 16S rDNA copies associated with a single A549 cell, which is directly proportional to the average number of contaminant per A549 cell. By using primers specific to the 16S rDNA sequence of the contaminant, we were able to estimate contaminants per single contaminated cell using both qPCR‐based relative and absolute quantification.