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Mycoplasma has long been recognised as a common contaminant of cell lines in continuous culture but their presence may go undetected for months. As the mycoplasma competes with the cell lines for the nutrients in culture media, one of the first signs is a reduction in the rate of cell proliferation and slight changes in cellular responses including gene expression, which leads to decreases in quantity or quality of product, inconsistency of manufacture, or possible adverse effects in biologic products. Until now, mycoplasma detection in cell cultures has been a long drawn out process with difficult to interpret results. The purpose of the present study is to describe the specificity of Bioluminescent assay (MycoAlertTM) as a new rapid alternative method for mycoplasma detection, i.e. culture method, PCR method, and staining method.
MycoAlertTM presented by Lonza, is a selective biochemical test that detects the activity of certain mycoplasmal enzymes. The viable mycoplasma are lysed and the enzymes react with the MycoAlertTM Substrate catalysing the conversion of ADP to ATP. By measuring the level of ATP in a sample both before and after the addition of MycoAlertTM Substrate, a ratio can be obtained which is indicative of the presence or absence of mycoplasma in the test sample. This simple test takes only 15 minutes to complete and its detection limit is approximately 50 cfu/ml.
In this study, we used mycoplasma species, M.arthritidis, M.hominis, M.fermentans, M.salivarium, M.orale, A.laidlawii, M.pirum are known to account for about 95% of the mycoplasma contamination in cell cultures. E. coli and P. aeruginosa that gram negative
bacteria, S. aureus and B. subtilis that gram positive bacteria used for negative control.
In the MycoAlertTM, the ratio of RLU were 14.79, 1.75, 25.95, 42.10, 97.36, 2.67 and 28.88
for each mycoplasma, respectively. All mycoplasma were positive. But the ratio of E.
coli, P. aeruginosa, S. aureus and B. subtilis were 0.18, 0.39, 0.37 and 0.38. The 4 species of bacteria were negative. For reference, in the PCR method, all mycoplasma samples were positive, but 4 species of bacteria were negative. In conclusion, we can make sure that the specificity of MycoAlertTM and understand it is a rapid, and easy method to detect mycoplasma compared to existing methods.