Institute of Molecular and Translational Medicine, Faculty of Medicine and Dentistry, Palacký University Olomouc, Hněvotínská 5, 779 00 Olomouc, Czech Republic.
Faculty of Chemical Technology, University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic.
Cells. 2019 Nov 25;8(12):1510. doi: 10.3390/cells8121510.
Contamination of cell cultures by mycoplasmas is a very common phenomenon. As they can substantially alter cell metabolism and potentially spread to all cell cultures in laboratory, their early detection is necessary. One of the fastest and cheapest methods of mycoplasma detection relies on the direct staining of mycoplasmas' DNA by DAPI or Hoechst dyes. Although this method is easy and fast to perform, it suffers from the low signal provided by these dyes compared to the nuclear DNA. Therefore, the reporter cell lines are used for cultivation of mycoplasmas before DAPI or the Hoechst staining step. In the study presented, we have developed and tested a new immunofluorescence assay for the detection of mycoplasmas. The method is based on the enzymatic labeling using DNA polymerase I and modified nucleotides utilizing nicks in the mycoplasmas' DNA. Modified nucleotides are incorporated into mycoplasmas' DNA and subsequently visualized by immunofluorescence microscopy. The developed approach is independent of the mycoplasma strain, does not intensely stain nuclear DNA, does not stain other bacteria, and provides higher sensitivity than the approach based on the direct labeling using DAPI or Hoechst dyes.
支原体污染细胞培养是一种非常常见的现象。由于它们可以极大地改变细胞代谢，并可能传播到实验室中的所有细胞培养物，因此需要及早发现。支原体检测最快、最便宜的方法之一是使用 DAPI 或 Hoechst 染料直接染色支原体 DNA。尽管这种方法易于操作且快速，但与核 DNA 相比，这些染料提供的信号较弱。因此，在 DAPI 或 Hoechst 染色步骤之前，使用报告细胞系培养支原体。在本研究中，我们开发并测试了一种新的免疫荧光检测方法来检测支原体。该方法基于利用 DNA 聚合酶 I 和修饰核苷酸对支原体 DNA 中的缺口进行酶标记。修饰核苷酸被掺入支原体 DNA 中，然后通过免疫荧光显微镜观察。所开发的方法不依赖于支原体株，不会强烈染色核 DNA，不会染色其他细菌，并且比基于 DAPI 或 Hoechst 染料的直接标记方法具有更高的灵敏度。
