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Demonstration of Mycoplasma Contamination in Cell Cultures by a Mycoplasma Group- Specific Polymerase Chain Reaction

  • Protocol
Human Cell Culture Protocols

Part of the book series: Methods in Molecular Medicine ((MIMM,volume 2))

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Abstract

Cell cultures are widely used in both medical and biotechnical research centers, industries, and also as diagnostic tools in a clinical setting It has been reported that up to 50% of cell cultures are contaminated with mycoplasmas (1). Mycoplasma contamination may alter cellular growth characteristics, enzyme patterns, and cell membrane composition, and can induce chromosomal abnormalities and cytopathogenic changes (2–5). In experimental results being published, it is now becoming standard practice to show that mycoplasma-free cell cultures have been used. For industrial production of biological materials derived from mammalian cells and intended for diagnostic or therapeutic use in humans, regulatory guidelines are emerging that are intended to guarantee the quality and safety of these products. Obligatory testing for mycoplasmas is therefore increasing (6)

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References

  1. Uphoff, C C, Brauer, S, Grunicke, D, Gignac, S M, Macleod, R A F, Quentmeier, H, Steube, K, Tummler, M., Voges, M., Wagner, and Drexler, H G (1992) Sensitivity and specificity of five different Mycoplasma detection assays Leukemia 6, 335–341.

    PubMed  CAS  Google Scholar 

  2. McGarrity, G J, Vanaman, V, and Sarama, J (1984) Cytogenetic effects of mycoplasma infection of cell cultures: a revtew In Vitro 20, 1–18

    Article  PubMed  CAS  Google Scholar 

  3. McGarrity, G J. and Kotani, H. (1985) Cell culture mycoplasmas, in The Mycoplasmas, vol IV (Razin, S and Barile, M F., eds), Academic, New York, pp 353–390

    Google Scholar 

  4. Barile, M. F (1979) Mycoplasma tissue cell culture interactions, in The Mycoplasmas (Tully, J G and Whitcomb, R F, eds.), Academic, New York, pp. 425–474

    Google Scholar 

  5. Stanbridge, E. J (1971) Mycoplasmas and cell cultures. Bacteriol Rev 35, 206–227

    PubMed  CAS  Google Scholar 

  6. Johansson, K.-E, Johansson, I, and Gobel, U B (1990) Evaluation of different hybridization procedures for the detection of mycoplasma contamination in cell cultures Mol Cell Probes 4, 33–42.

    Article  PubMed  CAS  Google Scholar 

  7. Del Giudice, R. A and Hopps, H. E. (1978) Microbiological methods and fluorescent microscopy for the direct demonstration of mycoplasma infection of cell cultures, in Mycoplasma Infections in Cell Cultures (McGarrity, G J., Murphy, D G, and Nichols, W W, eds), Plenum, New York, pp 57–69

    Google Scholar 

  8. Hopps, H E., Meyer, B C, Barile, M. F., and Del Giudice, R A (1973) Problems concerning “non-cultivable” mycoplasma contaminants in tissue cultures Ann NY Acad Sci 225, 265–276.

    Article  Google Scholar 

  9. Hay, R J., Macy, M. L, and Chen, T R (1989) Mycoplasma infection of cultured cells Nature 339, 487,488

    Article  PubMed  CAS  Google Scholar 

  10. McGarrity, G. J, Sarama, J, and Vanaman, V (1985) Cell culture techniques ASM News 51, 170–183

    Google Scholar 

  11. McGarrity, G J. (1982) Detection of mycoplasmal infection of cell cultures, in Advances in Cell Cultures (Maramorosch, K.), Academic, New York, pp. 99–131

    Google Scholar 

  12. Gobel, U B and Stanbridge, E. (1984) Cloned mycoplasma ribosomal RNA genes for the detection of mycoplasma contamination of tissue cultures Science 226, 1211–1213.

    Article  PubMed  CAS  Google Scholar 

  13. Razin, S, Gross, M., Wormser, M, Pollack, Y, and Glaser, G (1984) Detection of mycoplasmas Infecting cell cultures by DNA hybridization. In Vitro 20, 404–408.

    Article  PubMed  CAS  Google Scholar 

  14. Saiki, R K, Bugawan, T L., Horn, G. T., Mullis, K B, and Erlich, H A. (1986) Analysis of enzymatically amplified β-globin and HLA-DQ-alfa DNA with allelspecific oligonucleotide probes Nature 324, 163–166.

    Article  PubMed  CAS  Google Scholar 

  15. Blanchard, A., Gautier, M., and Mayau, V (1991) Detection and identification of mycoplasmas by amplification of rDNA. FEMS Microbiol Lett 81, 37–42

    CAS  Google Scholar 

  16. Spaepen, M., Angulo, A F, Marynen, P, and Cassiman, J.-J (1992) Detection of bacterial and mycoplasma contamination in cell cultures by polymerase chain reaction FEMS Microbiol Lett 99, 89–94

    Article  CAS  Google Scholar 

  17. Roulland-Dussoix, D., Henry, A, and Lemercier, B (1994) Detection of mycoplasmas in cell cultures by PCR. a one year study. J Microbiol. Methods 19, 27–134

    Google Scholar 

  18. Hopert, A, Uphoff, Wirth, M, Hauser, H., and Drexler, H. G. (1993) Specificity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines J Immunol Methods 164, 91–100.

    Article  PubMed  CAS  Google Scholar 

  19. Teyssou, R., Poutiers, F, Sarilard, C, Grau, O, Laigret, F, Bove, J-M, and Bebéar, C (1993) Detection of mollicute contamination in cell cultures by 16S rDNA amplification Mol Cell Probes 7, 209–216

    Article  PubMed  CAS  Google Scholar 

  20. van Kuppeveld, F J M, Johansson, K-E, Galama, J M. D, Kissing, J, Bolske, G, van der Logt, J T M, and Melchers, W J G (1994) Detection of Mycoplasma contamination in cell cultures by a Mycoplasma group-specific PCR Appl Environ Microbiol 60, 149–152

    PubMed  Google Scholar 

  21. Gray, M W, Sankoff, D, and Cedergren, R J (1984) On the evolutionary descent of organisms and organelles a global phylogeny based on highly conserved structural core in small subunit ribosomal RNA. Nucleic Acids Res. 12, 5837–5852

    Article  PubMed  CAS  Google Scholar 

  22. Waters, A P and McCutchan, T F (1990) Ribosomal RNA nature’s own polymerase-amplified target for diagnosis Parasitol Today 6, 56–59

    Article  PubMed  CAS  Google Scholar 

  23. van Kuppeveld, F J M, van der Logt, J T M, Angulo, A F, van Zoest, M J, Quint, W G V, Niesters, H G M, Galama, J M D, and Melchers, W J G (1992) Genus-and species-specific identification of mycoplasmas by 16S rRNA amplification Appl Environ Microbiol 58, 2606–2615 (Author’s correction [1993] 59, 655)

    PubMed  Google Scholar 

  24. Neefs, J-M, van der Peer, Y., Hendriks, L, and de Wachter, R (1990) Compilation of small ribosomal subunit RNA sequences Nucleic Acids Res 18, 2237–2318

    Article  PubMed  CAS  Google Scholar 

  25. van Kuppeveld, F J M, Melchers, W J G, Willemse, H, Kissing, J, Galama, J M D, and van der Logt, J T. M. (1993) Detection of Mycoplasma pulmonis in experimentally infected laboratory rats by 16S rRNA amplification J Clin Microbiol 31, 524–527

    PubMed  Google Scholar 

  26. Melchers, W J G, Verweij, P E, van den Hurk, P, van Belkum, A, De Pauw, E, Hoogkamp-Korstanje, J A. A, and Mets, J. F G M (1994) General primermediated PCR for detection of Aspergillus species. J Clin Microbiol 32, 1710–1717

    PubMed  CAS  Google Scholar 

  27. Kwok, S K (1990) Procedures to minimize PCR-product carry-over, in PCR Protocols A Guide to Methods and Applications (Innis, M A., Gelfand, D H, Sninsky, J J, and White, T J, eds), Academic, San Diego, C A, pp 142–145

    Google Scholar 

  28. Kwok, S and Higuchi, R (1989) Avoiding false positives with PCR Nature 339, 237,238

    Article  PubMed  CAS  Google Scholar 

  29. Southern, E M (1975) Detection of specific sequences among DNA fragments separated by gel electrophoresis J Mol Biol 98, 503–517

    Article  PubMed  CAS  Google Scholar 

  30. Ausubel, F M, Brent, R, Kingston, R E, Moore, D D., Seidman, J G., Smith, J A, and Struhl, K (1994) Current Protocols in Molecular Biology, vol I and II, Wiley, New York

    Google Scholar 

  31. Boehringer Mannheim Biochemica (1993) The DIG System User’s Guide for Filter Hybridization Boehringer Mannheim GmbH, Brochemica, Mannheim, Germany

    Google Scholar 

  32. Longo, M C, Berniger, M S, and Hartley, J L (1990) Use of uracil DNA glycosylase to control carry-over contamination in polymerase chain reactions Gene 93, 125–128

    Article  PubMed  CAS  Google Scholar 

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Author information

Authors and Affiliations

  1. Department of Medical Microbiology, University of Nijmegen, The Netherlands

    Frank van Kuppeveld & Jos van der Logt

  2. The National Veterinary Institute, Uppsala, Sweden

    Karl-Erik Johansson & Willem Melchers

Authors
  1. Frank van Kuppeveld
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  2. Jos van der Logt
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  3. Karl-Erik Johansson
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  4. Willem Melchers
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Editor information

Editors and Affiliations

  1. King’s College, University of London, UK

    Gareth E. Jones

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© 1996 Humana Press Inc., Totowa, NJ

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van Kuppeveld, F., van der Logt, J., Johansson, KE., Melchers, W. (1996). Demonstration of Mycoplasma Contamination in Cell Cultures by a Mycoplasma Group- Specific Polymerase Chain Reaction. In: Jones, G.E. (eds) Human Cell Culture Protocols. Methods in Molecular Medicine, vol 2. Humana Press. https://doi.org/10.1385/0-89603-335-X:525

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  • DOI: https://doi.org/10.1385/0-89603-335-X:525

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-335-1

  • Online ISBN: 978-1-59259-586-0

  • eBook Packages: Springer Protocols

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