A highly sensitive internally-controlled real-time PCR assay for mycoplasma detection in cell cultures

Mycoplasma contamination of cell lines is a common occurrence and may affect the cell line behaviour in a variety of ways. Contamination with mycoplasma is usually not obvious so cell lines should be frequently tested. Several commercially available kits for mycoplasma detection exist, however the Ph. Eur. culture method which can take several weeks to yield results is still considered to be the 'gold standard' method. There is therefore a need for rapid alternative methods with comparable sensitivity, specificity and species range. Here, we describe an internally-controlled Taqman-based real-time PCR assay for cell culture medium without the need for DNA extraction. The assay can detect less than 10 CFU of the most frequently encountered mycoplasma contaminants in mammalian cell cultures. The validated assay has the potential to be used as a routine test in the production of cell culture-based Biologicals.