Denis GERLIER - BIOBANQUES May 19, 2016
Biobanking National Infrastructure Meeting
Quality matters: Improving the quality of biological resources
Quantitative detection of Mycoplasma by Real-Time PCR of a 1.5 kb fragment using degenerate universal primers targeting 16S rDNA.
The ever-growing usage of cell lines to understand every biological process at the molecular and cellular level and the ability of microbes and parasites to invade them put these tools under stringent quality scrutiny to ensure unbiased interpretation of cell-based experiments. One of the major recognized pitfalls in cell culture is the adventitious contamination by Mollicutes, usually referred as mycoplasma, Mycoplasma being a major subgroup of the Mollicutes within the Tenericutes taxa. Several techniques have been developed to detect mycoplasma but they are either cumbersome, difficult to interpret and/or limited to the detection of only a limited range of species. We have adapted and extended a PCR that amplifies a long DNA fragment using universal degenerate primers so as to detect Mycoplasma 16S rDNA by Real-Time PCR (RT-PCR) followed by a sequencing-based identification step. We will report the advantages of this technique by comparison of three other methods, Hoechst DNA labelling, MycoAlert® and PlasmoTest® in detecting and identifying mycoplasma contamination in cell cultures including in BSL1 to BSL4 virus stocks. This work was supported by a grant from the French BioBanques Infrastructure, IBiSA and ANR.
THURSDAY MAY 19, 2016