Evaluation of mycoplasma removal reagents using qPCR-based quantification

In this study, we evaluated the efficacy of various mycoplasma removal reagents using nuclear staining, DNA gel electrophoresis, and qPCR-based quantification. Our results showed Plasmocure and Plasmocin are two effective anti-mycoplasma reagents whose effects can be observed within a week. However, prolonged treatment with Plasmocin led to development of resistance. Withdrawal of anti-mycoplasma reagents led to reoccurrence of mycoplasma contamination, but addition of prevention reagent, such as Primocin, prevented recontamination. Therefore, sequential treatment by Plasmocure and Primocin is the best course of action against mycoplasma contamination. Lastly, we developed methods based on qPCR to estimate the average number of mycoplasma associated with a single contaminated cell. We have shown, for the first time, that untreated contaminated BEAS-2B cells have approximately 300–400 mycoplasma contaminants per cell in the cytoplasm or attached to the cell membrane. Furthermore, withdrawal of anti-mycoplasma reagents led to reoccurrence of mycoplasma contamination within two days, and therefore continued use of prevention reagent is imperative. •Employed DNA gel electrophoresis and nuclear staining to preliminarily determine the level of mycoplasma contamination in cultured cells.•Assayed for five anti-mycoplasma reagents using DNA gel electrophoresis and nuclear staining and discovered an efficient and effective treatment using Plasmocure and Primocin.•Prolonged treatment with reagent containing single antibiotic, i.e. Plasmocin in this study, led to development of bacterial resistance and therefore should be avoided. Reagents containing more than one antibiotic or peptides are recommended.•Recontamination of mycoplasma is highly likely following withdrawal of reagents. Therefore, use of prevention reagent is mandatory.•qPCR-based quantification of mycoplasma in contaminated cultured cells was able to determine the number of mycoplasma in a single contaminated cell.