, Volume 61, Issue 3, pp 117–124 | Cite as

PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience

  • Vahid Molla Kazemiha
  • Mohammad Ali Shokrgozar
  • Mohammad Reza Arabestani
  • Morteza Shojaei Moghadam
  • Shahram Azari
  • Susan Maleki
  • Amir Amanzadeh
  • Mahmood Jeddi Tehrani
  • Fazel ShokriEmail author
Original Research


A total of 200 cell lines including different human, monkey, mice, hamster and rat cell types were examined for mycoplasma infection status. PCR assay using generic-specific universal primers showed that 40 (20%) of the cell lines are contaminated with mycoplasma. Employment of species-specific primers within these infected cell lines revealed infection with M. hyorhinis (42.5%), M. fermentas (37.5%), M. arginini (37.5%), M. orale (12.5%) and A. laidlawii (7.5%). A number of the cultures were coinfected with 2 or 3 different species. Contaminated samples were treated with BM-Cyclin, Ciprofloxacin and mycoplasma removal agent (MRA). Mycoplasma eradication was subsequently checked by PCR following 2 weeks continuous culture of treated cells in antibiotic free culture medium. Mycoplasmal infections were eradicated in 100, 70 and 42% of infected cell lines when the samples were treated with BM-Cyclin, MRA and Ciprofloxacin, respectively. However, 12% (BM-Cyclin), 62.5% (MRA) and 82.5% (Ciprofloxacin) of mycoplasma regrowth was observed 4 months after the treatment. Notably, the risk of spontaneous culture death was 17.5, 12.5 and 0% for BM-Cyclin, MRA and Ciprofloxacin, respectively.


Mycoplasmal infection Mammalian cell lines Elimination Antibiotics PCR-based detection 


  1. Barile MF, Rottem S (1993) Mycoplasma in cell culture. In: Kahane Ι, Adoni A (eds) Rapid diagnosis of mycoplasmas. Plenum Press, New York, pp 155–193Google Scholar
  2. Blanchard A, Montagnier L (1994) AIDS-associated mycoplasma. Annu Rev Microbiol 48:687–712CrossRefGoogle Scholar
  3. Boolske G (1988) Survey of mycoplasma in cell cultures and a comparison of detection methods. Zentralbl Bakteriol Mikrobiol Hyg 269:331–340Google Scholar
  4. Drexler HG, Uphoff CC (2002) Mycoplasma contamination of cell cultures: incidence, sources, effects, detection, elimination, prevention. Cytotechnology 39:75–90CrossRefGoogle Scholar
  5. Drexler HG, Dirks W, Macleod RAF et al (2001) DSMZ catalogue of human and animal cell lines, 8th edn. DSMZ, BraunschweigGoogle Scholar
  6. Drexler HG, Uphoff CC, Dirks WG et al (2002) Mix-ups and mycoplasma: the enemies within. Leuk Res 26:329–333CrossRefGoogle Scholar
  7. Fleckenstein E, Drexler HG (1996) Elimination of mycoplasma contamination in cell cultures. Biochemica 1:48–51Google Scholar
  8. Fleckenstein E, Uphoff CC, Drexler HG (1994) Effective treatment of mycoplasma contamination in cell lines with enrofloxacin (Baytril). Leukemia 8:1424–1434Google Scholar
  9. Gardella RS, Del Guidice RA (1984) Antibiotic sensitivities and elimination of mycoplasmas from infected cell cultures. Isr J Med Sci 20:931–934Google Scholar
  10. Gurbay A, Gontheir B, Barret L et al (2007) Cytotoxic effect of ciprofloxacin in primary culture of rat astrocytes and protection by vitamin E. Toxicology 229:54–61CrossRefGoogle Scholar
  11. Hopert A, Uphoff CC, Wirth M et al (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 Meth 164:91–100CrossRefGoogle Scholar
  12. Jung H, Wang SY, Ding WH et al (2003) Detection and treatment of mycoplasma contamination in cultured cells. Chang Gung Med J 26:250–258Google Scholar
  13. Kong F, James G, Gordon S et al (2001) Species-specific PCR for identification of common contaminant Mollicutes in cell culture. Appl Environ Microbiol 67:3195–3200CrossRefGoogle Scholar
  14. Kotani H, Butler G, Heggan D et al (1991) Elimination of mycoplasma from cell cultures by a novel soft agar technique. In Vitro Cell Dev Biol 27A:509–513CrossRefGoogle Scholar
  15. Lincoln CK, Lundin DJ (1990) Mycoplasma detection and control. US FCC Newsl 20:1–3Google Scholar
  16. Mattsson JG, Johansson KE (1993) Oligonucleotide probes complementary to 16S rRNA for rapid detection of mycoplasma contamination in cell cultures. FEMS Microbiol Lett 107:139–144CrossRefGoogle Scholar
  17. Razin S, Yogev D, Naot Y (1998) Molecular biology and pathogenicity of mycoplasma. Microbiol Mol Biol Rev 62:1094–1156Google Scholar
  18. Robinson LB, Wichelhausen RH (1956) Contamination of human cell cultures by pleuropneumonia like organisms. Science 124:1147–1148CrossRefGoogle Scholar
  19. Rottem S, Barile MF (1993) Beware of mycoplasma. Trends Biotechnol 11:143–151CrossRefGoogle Scholar
  20. Rottem S, Naot Y (1998) Subversion and exploitation of host cells by mycoplasmas. Trends Microbiol 6:436–440CrossRefGoogle Scholar
  21. Somasundaram C, Matzku S, Nickas W (1992) Use of ciprofloxacin and BM-Cyclin in mycoplasma decontamination. In Vitro Cell Dev Biol 28A:708–710CrossRefGoogle Scholar
  22. Tang J, Hu M, Lee S et al (2000) A polymerase chain reaction based method for detecting Mycoplasma/Acholeplasma contaminants in cell culture. J Microbial Methods 39:121–126CrossRefGoogle Scholar
  23. Timenetsky J, Santos L, Buzinhani M et al (2006) Detection of multiple mycoplasma infection in cell cultures by PCR. Braz J Med Biol Res 39:907–914CrossRefGoogle Scholar
  24. Uphoff CC, Drexler HG (2002) Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines. In Vitro Cell Dev Bio Anim 38:79–85CrossRefGoogle Scholar
  25. Uphoff CC, Gignac SM, Drexler HG (1992) Mycoplasma contamination in human leukemia cell lines. П. Elimination with various antibiotics. J Immunol Methods 149:55–62Google Scholar
  26. Uphoff CC, Meyer C, Drexler HG (2002) Elimination of mycoplasma from leukemia-lymphoma cell lines using antibiotics. Leukemia 16:284–288CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Vahid Molla Kazemiha
    • 1
  • Mohammad Ali Shokrgozar
    • 1
  • Mohammad Reza Arabestani
    • 1
  • Morteza Shojaei Moghadam
    • 2
  • Shahram Azari
    • 1
  • Susan Maleki
    • 3
  • Amir Amanzadeh
    • 1
  • Mahmood Jeddi Tehrani
    • 1
    • 4
    • 5
  • Fazel Shokri
    • 1
    • 6
    Email author
  1. 1.National Cell Bank of Iran, Pasteur Institute of IranTehranIran
  2. 2.Faculty of Biotechnology and Biomolecular SciencesUniversiti Putra MalaysiaUPM SerdangMalaysia
  3. 3.Department of Biology, School of ScienceShahid Chamran UniversityAhvazIran
  4. 4.Monoclonal Antibody Research CenterAvicenna Research InstituteTehranIran
  5. 5.Immune and Gene Therapy Laboratory, Cancer Center KarolinskaKarolinska InstituteStockholmSweden
  6. 6.Department of Immunology, School of Public HealthTehran University of Medical SciencesTehranIran

Personalised recommendations