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PCR Based Mycoplasma Detection

Mycoplasma, what is it?

Mycoplasma is a large group of tiny bacteria, about 0.3-0.8 um in diameter, all lacking a rigid cell wall belonging to the Mollicultes class of bacteria. Unlike other bacterial contaminates, the media will not change turbidity from a mycoplasma infection and therefore it will go undetected unless the culture is tested for mycoplasma. Due to the lack of a rigid cell wall and their small size, they are very difficult to eliminate once they contaminate a culture. Most antibiotics used in cell culturing, such as penicillin and streptomycin, work by attacking the rigid cell walls in bacteria, therefore since mycoplasma have no rigid cell wall they do not kill the mycoplasma and only mask the infection. The 0.3-0.8 um diameter and flexibility, from not having a rigid cell wall, allow these bacteria to pass through most of the conventional filters used in labs.

The two most common ways mycoplasma infection occurs: • Contamination from an already infected cell culture • Contamination from an infected lab member

Effects of mycoplasma on cultured cells: • Change in growth rate and metabolism • Change in cell morphology • Change in the composition of the cell membrane • Changes in gene expression • Chromosomal aberrations • Cell Death

How to prevent mycoplasma: • Always use Good Cell Culture Practice (including using anti-biotic free media when antibiotics are not necessary) • Quarantining all new cultures until mycoplasma testing has been completed • Regularly testing for mycoplasma

Mycoplasma PCR Based Detection Cell Line Genetics’ PCR based Mycoplasma detection is extremely sensitive, as it primes to the highly conserved 16S rRNA region of the mycoplasma’s genome. It detects 19 species of Mycoplasma and 1 species of Acholeplasma in any species of cells. Of those 19 species of mycoplasma, 6 are responsible for between 90-95% of all mycoplasma contamination in cultured stem cells.

  • Extremely sensitive PCR detects only the highly conserved 16s rRNA region
  • Detects 19 species of Mycoplasma and 1 species of Acholeplasma
  • Live cells or spent media passaged twice without antibiotics accepted
  • Fast turnaround time, highly accurate, competitively priced

References

1. Drexler, H. G., & Uphoff, C. C. (2002). Mycoplasma contamination of cell cultures: incidence, sources, effects, detection, elimination, prevention.Cytotechnology, 39(2), 75-90. 2. Uphoff, C. C., & Drexler, H. G. (2013). Detection of mycoplasma contaminations. In Basic Cell Culture Protocols (pp. 1-13). Humana Press. 3. Van Kuppeveld, F. J., Johansson, K. E., Galama, J. M., Kissing, J., Bölske, G., Van der Logt, J. T., & Melchers, W. J. (1994). Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR. Applied and environmental microbiology

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