PI-08. Detecting mycoplasma contamination of cell cultures by means of digital drop-PCR

Summary

Mycoplasma is a common contaminant of cell cultures. Small size and resistance to many antibiotics prevents their timely detection and elimination. Mycoplasma, exhausts the nutritional medium, inhibits the in vitro cell growth, alters distinct properties of cultured cells (metabolism, proliferation, gene expression) thus making it difficult to use them in experiments as model lines and interpret the study results. In this regard, it is extremely important to control the presence of this pathogen in laboratories. The method of digital drop PCR allows this assays at high specificity and reproducibility level thus making it preferable in the absence of alternative assays. The purpose of this work was to adapt droplet digital PCR technique for detection mycoplasma contamination of cell cultures, as well as to screen for mycoplasma the available human cell lines at the laboratories of R. Gorbacheva Research Institute.

Materials and methods

Within the framework of this study, we tested the K562, Raji, Hek293, THP-1 cell cultures with suspected contamination. Their aliquots were thawed and cultured in appropriate media for 48 hours prior to sampling. The cells were then washed of the culture medium by centrifugation and resuspension in phosphate-buffered saline. Further on, whole genome DNA was isolated from the cells using spin column isolation technology (GeneJETGenomic DNA PurificationKit, ThermoFisher, USA). Concentration and quality of DNA preparation was measured using a Nanodrop device. To detect mycoplasma contamination, the MycoReal-Time (Evrogen) reagent kit was used, with detection of mycoplasma DNA by the RT-PCR method with TaqMan probes, but with the addition of the ddPCR^TM SupermixforProbes reagent kit specific to the digital droplet PCR (Bio-Rad, USA). The droplets were generated, and fluorescence was read using a QX200 AutoDGDropletDigital PCR System (Bio-Rad, USA) with automatic droplet generation.

Results

In order to adapt the MycoReal-Time (Evrogen) protocol to digital droplet PCR, the following combination of reagents per reaction was used: 10 µl ddPCR^TM Supermix for Probes, 5 µl 5X OligoMyco RT, 2 µl cell line DNA sample or MycoDNA Control. To avoid an overload of DNA-containing droplets, positive control samples were diluted 4 times relative to those recommended by manufacturer. The total reaction volume was 25 µl. PCR was carried out as follows: 95°C, 10 min; (95°C, 10 sec; 60°C, 1 min)x45 cycles. Fluorescence values were read in the FAM channel. Using this protocol, a positive control signal from the MycoReal-Time kit (Eurogen) was successfully detected. Screening of laboratory cell lines showed that three out of four analyzed cell lines (K562, HEK2963T and THP-1) were contaminated with mycoplasma. An attempt to eliminate K562 contamination by culturing them in the presence of clindamycin (900 μg/ml) and ciprofloxacin (120 μg/ml) for 14 days was successful, i.e., mycoplasma was not detectable by the digital drop PCR technique.

Conclusion

According to the results of digital droplet PCR, the MycoReal-Time kit protocol (Evrogen) was successfully adapted to the QX200 AutoDGDropletDigital PCR System (Bio-Rad, USA). This technique may be used on routine basis in the laboratories of R. M. Gorbacheva Research Institute for the control of mycoplasma contamination of cultured cell lines and primary cultures of human cells.

Keywords

Mycoplasma, contamination, cell cultures, DNA, polymerase chain reaction (PCR), digital drop PCR.