Mycoplasma

Mycoplasmas are a large and widespread group of prokaryotes. This class presently comprises six eubacterial genera Acholeplasma, Anaeroplasma, Asteroplasma, Mycoplasma, Spiroplasma and Ureaplasma. A limited number of mycoplasma species can be found in 15-35% of all cell lines with predominantly human, swine, or bovine origin (Uphoff and Drexler, 2002). Over 95% of these are identified as Mycoplasma arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. pirum, and Acholeplasma laidlawii. Due to their small size (0.3-0.8 µm in diameter), they are the difficult to detect in culture and pose a main risk to experimental results. Due to their lack of a rigid wall they can pass through sterilization filter membranes. Mycoplasma contaminations affect many different aspects of the infected cell culture, leading to unreliable experimental results and possibly unsafe biological products. Therefore, testing for mycoplasma contamination is necessary to ensure reliable research results and quality biotechnological products. A typical finding of a Mycoplasma prevalence study stressed the importance of testing: 100% of the cultures from labs without mycoplasma testing programs were contaminated, but only 2% of the cultures from labs that tested regularly (McGarrity et al., 1986).

  

Effect on cell cultures

• Inhibition of cell proliferation by up to 50 % due to nutrition consumption and secretion of harmful catabolic products, inhibition of protein biosynthesis due to arginine depletion (McGarrity et al., 1984)
• Alteration of immunological reactions (macrophage activation, inhibition of antigen presentation, induction of signal transduction) (Mühlradt et al., 1996)
• Alteration of virus proliferation and infection rate
• Significant alteration of microarray gene expression profiles (Miller et al., 2003)
• Inhibition of hybridoma production, contaminated cells may become HAT-Media sensitive
• Adhesion of mycoplasma to cells alters cell wall integrity and affects membrane studies and patch-clamp experiments
• Mycoplasma can make up to 50 % of isolated proteins and 15-30 % of isolated DNA
• Reduction of transfection rate by electroporation down to 5 %

  

Detection by McCT

The primer sets used in McCT are directed to the highly conserved 16S rRNA coding region in the mycoplasma genome. This allows for amplification of all Mycoplasma and Acholeplasma species usually encountered as contaminants in cell cultures. Moreover, the McCT assay determines the genotype of Mycoplasma and Acholeplasma species by specific hybridisation, such as M. arginini, M. fermentans, M. orale, M. hyorhinis, M. hominis, M. salivarium, M. synoviae, M. pirum, M. gallisepticum, M. pneumoniae, and Acholeplasma laidlawii.