Mycoplasma contamination of cells is a widespread issue in many cell culture labs. It often goes unperceived due to the small size and slow replication of the mycoplasma. In addition, mycoplasma contaminations do not cause consistently observable alterations in cell culture. However, mycoplasmas contamination poses major threat on the reliability, reproducibility, and consistency of results in cell culture experiments. Mycoplasma can affect cellular functions in numerous ways including proliferation, protein synthesis, susceptibility to viral infection, etc. Hence rapid and accurate detection of mycoplasma is essential.

1st BASE Mycoplasma contamination is based on conventional (or endpoint) PCR, as the established method of choice for rapid, robust, and sensitive detection of mycoplasma contaminations. The primer set used is designed to specifically target and amplify the highly conserved 16S rRNA coding region of the mycoplasma genome. This allows detection of M. orale, M. hyorhinis, M. arginini, M. fermentans, M. salivarium, M. hominis, usually encountered as contaminants in cell cultures, as well as M. pneumoniae, Acholeplasma laidlawii, M. synoviae, Spiroplasma citri and Ureaplasma species.

Summary:

For accurate mycoplasma testing, samples should be collected from cell cultures at 80-90% confluence. Cell culture supernatants are ideal for testing and don't need additional preparation. However, PCR-inhibiting substances can accumulate in medium with over 12% serum, requiring DNA extraction before PCR. Mycoplasma titer in cell culture ranges from ~10^6 to 10^8 particles per ml, hence sufficient mycoplasma DNA is present in the supernatant for successful application of the PCR test.

Cell pellets and certain biological materials like fetal bovine serum, vaccines, cryo stocks, and paraffin-embedded samples must undergo extraction before PCR due to potential negative influences from cell debris.