, Volume 66, Issue 5, pp 861–873 | Cite as

Sensitivity of biochemical test in comparison with other methods for the detection of mycoplasma contamination in human and animal cell lines stored in the National Cell Bank of Iran

  • Vahid Molla Kazemiha
  • Amir Amanzadeh
  • Arash Memarnejadian
  • Shahram Azari
  • Mohammad Ali Shokrgozar
  • Reza Mahdian
  • Shahin Bonakdar
Original Research


Mycoplasma contamination in cell culture is considered as serious problem in the manufacturing of biological products. Our goal in this research is to find the best standard and rapid method with high sensitivity, specificity, accuracy and predictive values of positive and negative results for detection of mycoplasma contamination in cell cultures of the National Cell Bank of Iran. In this study, 40 cell lines suspected to mycoplasma contamination were evaluated by three different methods: microbial culture, enzymatic mycoalert® and molecular. Enzymatic evaluation was performed using the mycoalert® kit while in the molecular technique, a universal primer pair was designed based on the common and fixed 16SrRNA ribosomal sequences used. Mycoplasma contaminations in cell cultures with molecular, enzymatic and microbial culture methods were determined as 57.5, 52.5 and 40 %, respectively. These results confirmed the higher rate of sensitivity, specificity and accuracy for the molecular method in comparison with enzymatic and microbial methods. Polymerase chain reaction (PCR) assay based on fixed and common sequences in the 16SrRNA, is a useful valuable and reliable technique with high sensitivity, specificity and accuracy for detection of mycoplasma contamination in cell cultures and other biological products. The enzymatic mycoalert® method can be considered as a substitution for conventional microbial culture and DNA staining fluorochrome methods due to its higher sensitivity, specificity and speed of detection (<20 min).


Mycoalert® Cell culture Mycoplasma contamination Human-animal cell lines 



The authors would like to express their appreciation to Dr. Ehsan Mostafavi (head of Department of Epidemiology, Pasteur Institute of Iran) and also would like to thank National Cell Bank of Iran Pasteur Institute for their financial assistance.


  1. Cheng H-S, Shen C-W, Wang S-R (2007) Effect of storage conditions on detection of mycoplasma in biopharmaceutical products. In Vitro Cell Dev Biol Anim 43:113–119CrossRefGoogle Scholar
  2. Cheong KA, Agrawal SR, Lee AY (2011) Validation of nested PCR and a selective biochemical method as alternatives for mycoplasma detection. J Basic Microbiol 51:215–219CrossRefGoogle Scholar
  3. Dabrazhynetskaya A, Volokhov D, David S, Ikonomi P, Brewer A, Chang A, Chizhikov V (2011) Preparation of reference strains for validation and comparison of mycoplasma testing methods. J Appl Microbiol 111:904–914CrossRefGoogle Scholar
  4. Del Giudice R, Gardella R, Hopps H (1980) Cultivation of formerly noncultivable strains ofMycoplasma hyorhinis. Curr Microbiol 4:75–80CrossRefGoogle Scholar
  5. Freshney RI, Vunjak-Novakovic G, Freshney R (2006) Basic principles of cell culture. Cult Cells Tissue Eng 7:11–14Google Scholar
  6. Galen RS, Gambino SR (1975) Beyond normality: the predictive value and efficiency of medical diagnoses. Wiley, New YorkGoogle Scholar
  7. Garner C, Hubbold L, Chakraborti P (2000) Mycoplasma detection in cell cultures: a comparison of four methods. Br J Biomed Sci 57:295Google Scholar
  8. Harasawa R, Mizusawa H, Fujii M, Yamamoto J, Mukai H, Uemori T, Asada K, Kato I (2005) Rapid detection and differentiation of the major mycoplasma contaminants in cell cultures using real-time PCR with SYBR Green I and melting curve analysis. Microbiol Immunol 49:859–863CrossRefGoogle Scholar
  9. Harlin H, Gajewski TF (2008) Diagnosis and treatment of mycoplasma-contaminated cell cultures. Curr Protocol Cytom 43:A. 3C. 1–A. 3C. 7Google Scholar
  10. Hay RJ, Ikonomi P (2005) Detection of microbial and viral contaminants in cell lines. In: Cell biology, vol 1, p 49.Google Scholar
  11. Hong S, Lee H-A, Park S-H, Kim O (2011) Sensitive and specific detection of mycoplasma species by consensus polymerase chain reaction and dot blot hybridization. Lab Anim Res 27:141–145CrossRefGoogle Scholar
  12. Hopert A, Uphoff CC, Wirth M, Hauser H, Drexler HG (1993) Specifity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines. J Immunol Methods 164:91–100CrossRefGoogle Scholar
  13. Huang SM, Strong JM, Zhang L, Reynolds KS, Nallani S, Temple R, Abraham S, Habet SA, Baweja RK, Burckart GJ (2008) New era in drug interaction evaluation: US Food and Drug Administration update on CYP enzymes, transporters, and the guidance process. J Clin Pharmacol 48:662–670CrossRefGoogle Scholar
  14. Jung H, Wang SY, Yang IW, Hsueh DW, Yang WJ, Wang TH, Wang HS (2003) Detection and treatment of mycoplasma contamination in cultured cells. Chang Gung Med J 26:250–258Google Scholar
  15. Kong H, Volokhov DV, George J, Ikonomi P, Chandler D, Anderson C, Chizhikov V (2007) Application of cell culture enrichment for improving the sensitivity of mycoplasma detection methods based on nucleic acid amplification technology (NAT). Appl Microbiol Biotechnol 77:223–232CrossRefGoogle Scholar
  16. Lawrence B, Bashiri H, Dehghani H (2010) Cross comparison of rapid mycoplasma detection platforms. Biologicals 38:218–223CrossRefGoogle Scholar
  17. Mariotti E, Mirabelli P, Di Noto R, Fortunato G, Salvatore F (2008) Rapid detection of mycoplasma in continuous cell lines using a selective biochemical test. Leuk Res 32:323–326CrossRefGoogle Scholar
  18. Markoullis K, Bulian D, Hölzlwimmer G, Quintanilla-Martinez L, Heiliger K-J, Zitzelsberger H, Scherb H, Mysliwietz J, Uphoff CC, Drexler HG (2009) Mycoplasma contamination of murine embryonic stem cells affects cell parameters, germline transmission and chimeric progeny. Transgenic Res 18:71–87CrossRefGoogle Scholar
  19. McGarrity GJ, Kotani H (1985) Cell culture mycoplasmas. In: Razin S, Barile MF (eds) The Mycoplasmas, vol 4, Academic Press Inc., New York, pp 353–390Google Scholar
  20. McGarrity G, Sarama J, Vanaman V (1985) Cell culture techniques. ASM News 51:170–183Google Scholar
  21. Molla Kazemiha V, Shokrgozar MA, Arabestani MR, Moghadam MS, Azari S, Maleki S, Amanzadeh A, Tehrani MJ, Shokri F (2009) PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience. Cytotechnology 61:117–124CrossRefGoogle Scholar
  22. Molla Kazemiha V, Azari S, Amanzadeh A, Bonakdar S, Moghadam MS, Anbouhi MH, Maleki S, Ahmadi N, Mousavi T, Shokrgozar MA (2011) Efficiency of Plasmocin™ on various mammalian cell lines infected by mollicutes in comparison with commonly used antibiotics in cell culture: a local experience. Cytotechnology 63:609–620CrossRefGoogle Scholar
  23. Nikfarjam L, Farzaneh P (2012) Prevention and detection of mycoplasma contamination in cell culture. Cell J 13:203–212Google Scholar
  24. Peredeltchouk M, Wilson David S, Bhattacharya B, Volokhov D, Chizhikov V (2011) Detection of mycoplasma contamination in cell substrates using reverse transcription PCR assays. J Appl Microbiol 110:54–60CrossRefGoogle Scholar
  25. Pitt A, Crouch SPM, Slater KJ, Cox A (2012) Assay for detecting mycoplasma by measuring acetate kinase or carbamate kinase activity. European Patent No. EP 2264181Google Scholar
  26. Puppe W, Weigl J, Gröndahl B, Knuf M, Rockahr S, von Bismarck P, Aron G, Niesters H, Osterhaus A, Schmitt H-J (2013) Validation of a multiplex reverse transcriptase PCR ELISA for the detection of 19 respiratory tract pathogens. Infection 41:77–91CrossRefGoogle Scholar
  27. Robinson LB, Wichelhausen RH, Roizman B (1956) Contamination of human cell cultures by pleuro pneumonia like organisms. Science 124:1147–1148Google Scholar
  28. Razin S, Yogev D, Naot Y (1998) Molecular biology and pathogenicity of mycoplasmas. Microbiol Mol Biol Rev 62:1094–1156Google Scholar
  29. Smith A, Mowles J (1996) Prevention and control of mycoplasma infection of cell cultures. In: Tully JG, Razin S (eds) Molecular and diagnostic procedures in mycoplasmology, Academic Press, San Diego pp 445–451Google Scholar
  30. Störmer M, Vollmer T, Henrich B, Kleesiek K, Dreier J (2009) Broad-range real-time PCR assay for the rapid identification of cell-line contaminants and clinically important mollicute species. Int J Med Microbiol 299:291–300CrossRefGoogle Scholar
  31. Tang J, Hu M, Lee S, Roblin R (2000) A polymerase chain reaction based method for detecting Mycoplasma Acholeplasma contaminants in cell culture. J Microbiol Methods 39:121–126CrossRefGoogle Scholar
  32. Timenetsky J, Santos L, Buzinhani M, Mettifogo E (2006) Detection of multiple mycoplasma infection in cell cultures by PCR. Braz J Med Biol Res 39:907–914CrossRefGoogle Scholar
  33. Uphoff CC, Drexler HG (2002) Comparative PCR analysis for detection of mycoplasma infections in continuous cell lines. In Vitro Cell Dev Biol Anim 38:79–85Google Scholar
  34. Uphoff CC, Drexler HG (2011) Detecting Mycoplasma contamination in cell cultures by polymerase chain reaction. In: Cree IA (ed) Cancer cell culture. Humana Press, Chicago, pp 93–103Google Scholar
  35. Uphoff CC, Drexler HG (2013) Detection of mycoplasma contaminations. In: Helgason CD, Miller CL (eds) Basic cell culture protocols. Humana Press, Chicago, pp 1–13Google Scholar
  36. Uphoff CC, Gignac SM, Drexler HG (1992) Mycoplasma contamination in human leukemia cell lines. I. Comparison of various detection methods. J Immunol Methods 149:43–53CrossRefGoogle Scholar
  37. Volokhov DV, Kong H, George J, Anderson C, Chizhikov VE (2008) Biological enrichment of Mycoplasma agents by cocultivation with permissive cell cultures. Appl Environ Microbiol 74:5383–5391CrossRefGoogle Scholar
  38. Volokhov DV, Graham LJ, Brorson KA, Chizhikov VE (2011) Mycoplasma testing of cell substrates and biologics: review of alternative non-microbiological techniques. Mol Cell Probes 25:69–77CrossRefGoogle Scholar
  39. Waites KB, Katz B, Schelonka RL (2005) Mycoplasmas and ureaplasmas as neonatal pathogens. Clin Microbiol Rev 18:757–789CrossRefGoogle Scholar
  40. Yavlovich A, Kohen R, Ginsburg I, Rottem S (2006) The reducing antioxidant capacity of Mycoplasma fermentans. FEMS Microbiol Lett 259:195–200CrossRefGoogle Scholar
  41. Young L, Sung J, Stacey G, Masters JR (2010) Detection of Mycoplasma in cell cultures. Nat Protoc 5:929–934CrossRefGoogle Scholar
  42. Zhang S, Tsai S, Lo S-C (2006) Alteration of gene expression profiles during mycoplasma-induced malignant cell transformation. BMC Cancer 6:116CrossRefGoogle Scholar
  43. Zhao H, Dreses-Werringloer U, Davies P, Marambaud P (2008) Amyloid-beta peptide degradation in cell cultures by mycoplasma contaminants. BMC Res Notes 1:38CrossRefGoogle Scholar
  44. Zhi Y, Mayhew A, Seng N, Takle GB (2010) Validation of a PCR method for the detection of mycoplasmas according to European Pharmacopoeia section 2.6. 7. Biologicals 38:232–237CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Vahid Molla Kazemiha
    • 1
  • Amir Amanzadeh
    • 1
  • Arash Memarnejadian
    • 2
  • Shahram Azari
    • 1
  • Mohammad Ali Shokrgozar
    • 1
  • Reza Mahdian
    • 3
  • Shahin Bonakdar
    • 1
  1. 1.National Cell Bank of IranPasteur Institute of IranTehranIran
  2. 2.Department of Hepatitis and AIDSPasteur Institute of IranTehranIran
  3. 3.Molecular Medicine Group, Department of BiotechnologyPasteur Institute of IranTehranIran

Personalised recommendations