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Mycoplasma detection to confirm mycoplasma contamination (CAT#: STEM-CBT-0035-WXH)
Introduction
Mycoplasma corrupts cell cultures easily—and without scientists’ knowledge—for a multitude of reasons. Mycoplasma doesn’t cause media turbidity or pH changes, brings about few metabolic byproducts, and can’t be detected by microscopy. Once it has contaminated cells, mycoplasma can cause chromosomal aberrations, disrupt nucleic acid synthesis, change membrane antigenicity, inhibit cell proliferation and metabolism, decrease transfection rates, change gene expression profiles, and even bring about cell death. For these reasons, routine screening of cell lines for mycoplasma contamination is essential. The only way to confirm mycoplasma contamination is by routine testing using one or more special techniques, including direct growth on broth/agar, specific DNA staining, PCR, ELISA, RNA labeling and enzymatic procedures.
Principle
Take PCR method as example:
Mycoplasma DNA in the sample can be detected by PCR. The amplified gene codes for 16S ribosomal RNA, and primers have been selected so that all Mycoplasma ssp, Ureaplasma ssp, Spiroplasma spp, and Acholeplasma spp. are detected. The detection limit is < 50 bacteria for all examined species. The validity of a negative result is ensured by simultaneous amplification of an internal amplification control.
Mycoplasma DNA in the sample can be detected by PCR. The amplified gene codes for 16S ribosomal RNA, and primers have been selected so that all Mycoplasma ssp, Ureaplasma ssp, Spiroplasma spp, and Acholeplasma spp. are detected. The detection limit is < 50 bacteria for all examined species. The validity of a negative result is ensured by simultaneous amplification of an internal amplification control.
Applications
Confirm mycoplasma contamination.
Procedure
• Culture isolation method
The so-called gold standard test for the most accurate detection of mycoplasma is the culture isolation method, also known as the agar and broth procedure.
• DNA detection method
Cells are stained for DNA with a fluorescent dye (such as Hoechst dye), and mycoplasmas that had been in your cell sample will be visible as fluorescent spots, or granules, surrounding the nucleus of the indicator cells.
• PCR method method
Perform PCR on samples of your suspected cell cultures, using primers specific for mycoplasma DNA, usually mycoplasma’s 16S rRNA genes. Running the PCR product on a gel shows the presence of mycoplasma DNA by band(s) of distinct sizes.
• Enzymatic and ELISA method
Substrate converts ADP to ATP in the presence of mycoplasmal enzymes. The test reagent also contains another compound such as the enzyme luciferase which uses the ATP to produce light-signaling the presence of mycoplasma infection in your cultures.
The so-called gold standard test for the most accurate detection of mycoplasma is the culture isolation method, also known as the agar and broth procedure.
• DNA detection method
Cells are stained for DNA with a fluorescent dye (such as Hoechst dye), and mycoplasmas that had been in your cell sample will be visible as fluorescent spots, or granules, surrounding the nucleus of the indicator cells.
• PCR method method
Perform PCR on samples of your suspected cell cultures, using primers specific for mycoplasma DNA, usually mycoplasma’s 16S rRNA genes. Running the PCR product on a gel shows the presence of mycoplasma DNA by band(s) of distinct sizes.
• Enzymatic and ELISA method
Substrate converts ADP to ATP in the presence of mycoplasmal enzymes. The test reagent also contains another compound such as the enzyme luciferase which uses the ATP to produce light-signaling the presence of mycoplasma infection in your cultures.
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For various laboratory needs
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