by akronbiotech

 

Mycoplasma contamination of cell cultures is an alarming reality: 25% of all cultures are reported to be contaminated with Mycoplasma, and further challenges – such as the fact Mycoplasma is hard to detect with regular light microscopes – make these numbers even more urgent. Mycoplasmas are very small prokaryotes which lack a cell wall.

The best option for mycoplasma-infected cell cultures remains to discard these cultures and replace them with mycoplasma-free, fresh stocks. Though different elimination techniques have appeared, because such a drastic outcome can lead to sever consequences for sensitive experimental workflows, efficient and rapid tests for Mycoplasma detection remain a necessity. Especially considering the potential cost of research material lost to mycoplasma-contaminated cultures: hundreds of millions of dollars in research funding are potentially affected, and likely negatively compromised, due to mycoplasma contamination.

Mycoplasma detection techniques are not new, though they continue to be developed and studied. Culture-based mycoplasma detection methods are the gold standard techniques used to detect mycoplasma contamination in cultures. They are, however, slow and cumbersome, and do not meet the needs for new generations of cell therapies, where immediacy is critical. As an alternative, PCR or RT-PCR detection methods have emerged as robust, and rapid, alternatives.

In a new study by the lab of Denis Gerlier at the International Center for Infectiology Research at the University Claude Bernard in Lyon, tested mycoplasma detection by real-time PCR which they called m16S_qPCR. The technique is based on the selective amplification of a 1.5 kilobase DNA fragment using universal degenerate U1/U8 primers that target the mycoplasma 16S rDNA. To validate the method, the authors used hundreds of samples from either cell culture or BSL2 to BSL4 viral stocks and compared them to non-RT-PCR-based commercial methods, including Hoechst DNA staining, MycoAlert and PlasmoTest and PCR.

The article, titled Assessing mycoplasma contamination of cell cultures by qPCR using a set of universal primer pairs targeting a 1.5 kb fragment of 16S rRNA genes, was published recently in PLoS One.

Sensitivity results are shown in the figure below.

 

Sensitivity and specificity of five mycoplasma detection methods. From Jean et al, PLoS One 2017.

The authors found that m16S_qPCR procedure has significant advantage over alternative methods for Mycoplasma detection: a very broad coverage, high sensitivity (>19 16S rDNA copies), the incorporation of a DNA loading probe, and the use of a traceable positive reference control.

The study further demonstrates that RT-PCR-based methods will likely grow in interest as speed, accuracy and sensitivity become more important as new cell therapies reach advanced clinical stages.

One such method is already available to try out. Akron’s Mycosolutions Mycoplasma Real-Time PCR Detection Kit is an easy-to-use kit that allows a fast and highly sensitive detection of Mycoplasma contamination in biological samples. The primers are specific to a segment of the 16S-23S rRNA region of the Mycoplasma genome, which is highly conserved within the species Mycoplasma.

The primers of the kit were tested for years in comparison to the culture detection method and found to be very reliable and specific to Mycoplasma, while not reacting with mammalian or bacterial cells. The primers have broad detection range – more than 95% of the Mycoplasma species found in cell culture.

If you want to try out the kit, please contact us.

 



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