Did you know that almost up to 35% of continuous cell lines have mycoplasma contamination according to an article where mycoplasma incidence was compared in different cell lines?
Mycoplasma is not only a problem in old cell lines, but it can be present in new cell lines and even primary cells. Even when the cells are cultured with antibiotics, mycoplasma starts to easily take over cells. It is important to test cell cultures regularly to ensure reproducibility of results during projects and experiments. Mycoplasma is known to interfere for instance with levels of RNA, DNA and protein synthesis, signal transduction and induction of lymphocyte activation. (1)
Mycoplasma can be tested with three easy methods – PCR, luminescence or visual color change based assay. All three methods are fast and easy to perform. To test cell cultures for mycoplasma, they need to grow in the same culturing media for few days before taking the sample.
Detecting mycoplasma with PCR (results in 5 h, hands on 20 min)
The advantage of using PCR based assay to detect mycoplasma is that it only requires two common laboratory methods, PCR and gel electrophoresis, that are routine thing in almost all laboratories. The test can be simply performed by running a PCR with a kit containing all the needed primers and other components and then running it on an agarose gel to detect if any of the mycoplasma specific genes were copied. Running PCR and gel is slightly more time consuming than the methods based on luminescence, but the benefit is that it only requires PCR machine and a gel run and the process can be speeded up by using a ready-to-use master mix specifically designed for mycoplasma detection.
Recommended PCR based test: N-GARDE Mycoplasma PCR Reagent set (Euroclone) EMK090020.
Detecting mycoplasma with luminescence (results in 20 min)
When a simple yes or no is enough accuracy for mycoplasma test, luminescence based methods are faster to perform. A good example is Lonza’s MycoAlert™ kit. A selective biochemical test exploits the activity of mycoplasmal enzymes which are found in all six of the main mycoplasma cell culture contaminants and the vast majority of 180 mycoplasma species, but are not present in eukaryotic cells. The lysed cells are reacting with the MycoAlert™ substrate, which catalyzes the conversion of ADP to ATP, which in turns activates the luciferase enzyme leading to catalyzation of light signal. The method requires a tube or plate luminometer.
Recommended luminescence based test: MycoAlert PLUS detection kit (Lonza) LT07-703.
Detecting mycoplasma with bare eye (results in 60 to 90 min)
Mycoplasma test can be performed even when there is no possibility to use a PCR machine or luminescence reader. AssayGenie has developed an easy and accurate method to detect mycoplasma that is based on enzyme that changes the color of the sample mix depending on the presence or absence of mycoplasma. Sky blue color is a sign of mycoplasma contamination whereas purple means mycoplasma free sample. The method is as accurate as homebrew qPCR methods, but faster to perform. The kit includes a positive control. For more detailed information and a protocol visit: https://www.assaygenie.com/mycogenie-rapid-mycoplasma-detection-kit/.
Recommended non-instrument based test: MycoGenie Rapid Mycoplasma Detection kit (AssayGenie) MORV0011.
 Drexler HG, Uphoff CC. Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention. Cytotechnology. 2002 Jul;39(2):75-90. doi: 10.1023/A:1022913015916. PMID: 19003295; PMCID: PMC3463982.
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