Possibilities of qPCR control of mycoplasma contamination of cell cultures
Abstract
Mycoplasmas are the main contaminants of cells cultures. They persist in cell cultures and can extensively affect host cell functions. Conducting experiments or producing protein in contaminated cultures are impractical. The aim of the study was to explore the possibility of quick detection of mycoplasma contamination of cell cultures and biotechnological products by a previously developed method which was modified to make it simpler and more affordable in Russia; and to assess the possibility of method validation for quality control. The authors chose the most applicable qPCR method of all qPCR methods currently used for mycoplasma detection, and modified it in the following way: expensive MGB-probes which cannot be synthesized in Russia were substituted by ordinary fluorescence probes. The reproducibility and sensitivity of the modified method were tested with M. hominis. The sensitivity of the test was equal to 10 mycoplasma gene copies per reaction. Comparison of the obtained results with regulatory requirements for mycoplasma detection showed that the proposed method complies with current official requirements and could be used as the main method for routine prompt cell culture testing for mycoplasma contamination.
About the Authors
N. D. Yolshin
State Research Institute of Highly Pure Biopreparations
Russian Federation
Junior research scientist of the Immunopharmacology Laboratory
Russian Federation
Junior research scientist of the Immunopharmacology Laboratory
A. V. Petrov
State Research Institute of Highly Pure Biopreparations
Russian Federation
Head of the Immunopharmacology Laboratory
Russian Federation
Head of the Immunopharmacology Laboratory
References
1. Langdon SP. Cell culture contamination: an overview. Methods Mol Med. 2004; 88: 309–17.
2. Uphoff CC, Denkmann SA, Drexler HG. Treatment of mycoplasma contamination in cell cultures with Plasmocin. J Biomed Biotechnol. 2012; 2012: 267678.
3. Rottem S, Barile MF. Beware of mycoplasmas. Trends Biotechnol. 1993; 11(4): 143–51.
4. Stemke GW, Robertson JA. Comparison of two methods for enumeration of mycoplasmas. J Clin Microbiol. 1982; 16(5): 959–61.
5. Olarerin-George AO, Hogenesch JB. Assessing the prevalence of mycoplasma contamination in cell culture via a survey of NCBI’s RNA-seq archive. Nucleic Acids Res. 2015; 43(5): 2535–42.
6. van Kuppeveld FJ, van der Logt JT, Angulo AF, van Zoest MJ, Quint WG, Niesters HG, et al. Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl Environ Microbiol. 1992; 58(8): 2606–15.
7. Lincoln CK, Gabridge MG. Cell culture contamination: sources, consequences, prevention, and elimination. Methods Cell Biol. 1998; 57: 49–65.
8. OFS. 1.7.2.0031.15. Testing for the presence of mycoplasma. State Pharmacopoeia of the Russian Federation. 13th ed. V. 2. P. 827–35. Available from: http://www.femb.ru/feml.
9. 2.6.7. Mycoplasmas. European Pharmacopoeia 9.0. Available from: http://pharmeuropa.edqm.eu/ep900.
10. Volokhov DV, Graham LJ, Brorson KA, Chizhikov VE. Mycoplasma testing of cell substrates and biologics: Review of alternative non-microbiological techniques. Mol Cell Probes 2011; 25(2 – 3): 69–77.
11. Yolshin ND. Detection of residual E. coli and CHO host-cell DNA in recombinant proteins by qPCR. BIOpreparations. Prevention, Diagnosis, Treatment 2016; 16(4): 245–52 (in Russian).
12. Janetzko K, Rink G, Hecker A, Bieback K, Kluter H, Bugert P. A single-tube real-time PCR assay for Mycoplasma detection as a routine quality control of cell therapeutics. Transfus Med Hemother. 2014; 41(1): 83–9.
13. 1st WHO International Standard for mycoplasma DNA for nucleic acid amplification technique-based assays designed for generic mycoplasma detection. Available from: https://goo.gl/Ue2xP9.
14. Kutyavin IV, Afonina IA, Mills A, Gorn VV, Lukhtanov EA, Belousov ES, et al. 3’-minor groove binder-DNA probes increase sequence specificity at PCR extension temperatures. Nucleic Acids Res. 2000; 28(2): 655–61.
15. Shalunova NV, Volkova RA, Volgin AR, Petruchuk EM, Berdnikova ZE, Elbert EV, et al. Mycoplasma contamination of cell cultures. BIOpreparations. Prevention, Diagnosis, Treatment 2016; 16(3): 151–60 (in Russian).
16. Peredeltchouk M, David SA, Bhattacharya B, Volokhov DV, Chizhikov V. Detection of mycoplasma contamination in cell substrates using reverse transcription-PCR assays. J Appl Microbiol. 2011; 110(1): 54–60.
Review
For citations:
Yolshin N.D., Petrov A.V. Possibilities of qPCR control of mycoplasma contamination of cell cultures. BIOpreparations. Prevention, Diagnosis, Treatment. 2017;17(3):173-179. (In Russ.)
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