BACKGROUND:
Mycoplasmas are very small free-living organisms similar to bacteria but lacking a cell wall. They are ubiquitous in nature and are potential contaminants in biotechnology and cell-based products. There are several detection methods available, including conventional culture enrichment and growth on agar, cell culture substrate with Hoechst staining and more rapid Polymerase Chain Reaction (
PCR) methods. Biological products thus require examination for presence/absence of mycoplasmas before release for sale/use. Since mycoplasmas have been commonly found to contaminate cell cultures, it is also wise to check these periodically to ensure they remain free from infection. This is especially important where these cells may be developed as Master Cell stock for product development purposes.
AMS Laboratories are able to offer this detection service.
PRINCIPLES:
Mycoplasma detection by conventional test methods
Two test methods are followed to detect Mycoplasma contamination:
- Enrichment in broth and detection by colony formation on selective agar plates. Samples are set up as per pharmacopoeia method whereby product is inoculated into mycoplasma enrichment broth and onto selective medium agar plates. Broths are sub-cultured onto further agar plates for up to two weeks. Cultures are incubated aerobically and microaerophilically. Inoculated media are examined for evidence of mycoplasma growth.
-
Enrichment in cell culture and characteristic fluorescent staining of
DNA. Samples are inoculated onto susceptible mycoplasma-free host cell cultures and incubated for 4 days. Cells are then passaged to fresh cultures and further incubated. After incubation, cells are stained with Hoechst stain and examined under a fluorescent microscope for signs of mycoplasma infection.
PCR test for Mycoplasma detection
A more rapid detection method for mycoplasmas is available through the use of the PCR technique. This has recently been added to the European Pharmacopoeia as an approved method as well as the conventional methods like the broth/agar plate enrichment method and the indicator cell culture method.
The basic principle is the amplification of the Mycoplasmal
DNA from within the sample matrix using specifically designed primers. These are then detected under UV illumination in a G-box.
The biggest advantages of this method are:
- Simplicity and Reliability
- Robustness
-
Rapidity of the test
SAMPLE REQUIREMENTS and TURNAROUND TIMES:
Pls contact the laboratory to discuss your project requirements |
