Abstract
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.
Highlights
Mycoplasmas are wall-less bacteria [1]
The mycoplasma infection was confirmed by room temperature (RT)-polymerase chain reaction (PCR), the developed approach (Supplementary Figure S1a,b), and a MycoAlertTM PLUS Mycoplasma Detection Kit (East Port Praha s r. o. (Lonza, LT07-703), Prague, Czech Republic)
Brief washing steps using 1× PBS buffer or Tris-NaCl buffer are inserted among the particular steps
Summary
Mycoplasmas are wall-less bacteria [1]. It is presumed that the lack of the cell wall and of some enzymatic activities and metabolic pathways is the result of degenerative evolution. In the case of the agar/broth media assay, only cultivable mycoplasmas are detected (mycoplasmas able to grow on the used medium). Mycoplasmas’ DNA is stained using DAPI of Hoechst dyes [2,4,6] The disadvantage of both approaches is the long testing time (circa 28 days in the case of the agar/broth media assay and circa 10 days in the case of the indicator cell culture assay). Contrary to the method based on DAPI or Hoechst staining, the developed method does not intensively label nuclear DNA and enables users to choose the fluorochrome to match their needs and it allows a much clearer interpretation of the obtained results, in the case of the low density of mycoplasmas
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