Skip to main content
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1996 Feb;62(2):328–331. doi: 10.1128/aem.62.2.328-331.1996

Application of a Mycoplasma group-specific PCR for monitoring decontamination of Mycoplasma-infected Chlamydia sp. strains.

J M Ossewaarde 1, A de Vries 1, T Bestebroer 1, A F Angulo 1
PMCID: PMC167802  PMID: 8593037

Abstract

Mycoplasma contamination of biological materials remains a major problem. Most contaminations are caused by the use of Mycoplasma-contaminated cell lines. We adapted a Mycoplasma group-specific PCR to detect Mycoplasma contamination in cell lines and demonstrate its use in monitoring decontamination procedures with Mycoplasma-contaminated suspensions of Chlamydia spp. Three different methods were investigated: the use of Mycoplasma-specific antiserum in cell culture, physical separation by the combined use of enzymatic treatment and differential centrifugation, and the use of detergents. With these methods only incubation with Triton X-100 resulted in decontamination of Mycoplasma-contaminated suspensions of several laboratory strains of Chlamydia pneumoniae, C. pecorum, and C. trachomatis. Only one C. pneumoniae strain, UZG-1, was sensitive to Triton X-100 treatment. Since 39 of 40 throat swabs from patients with symptoms of an upper respiratory tract infection had positive reactions in the Mycoplasma group-specific PCR, this procedure could also have clinical significance in attempts to propagate C. pneumoniae strains from clinical specimens.

Full Text

The Full Text of this article is available as a PDF (221.3 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Altschul S. F., Gish W., Miller W., Myers E. W., Lipman D. J. Basic local alignment search tool. J Mol Biol. 1990 Oct 5;215(3):403–410. doi: 10.1016/S0022-2836(05)80360-2. [DOI] [PubMed] [Google Scholar]
  2. Carroll K., O'Kennedy R. The elimination of mycoplasma from infected hybridomas by passaging in BALB/c mice. J Immunol Methods. 1988 Apr 6;108(1-2):189–193. doi: 10.1016/0022-1759(88)90418-8. [DOI] [PubMed] [Google Scholar]
  3. Don R. H., Cox P. T., Wainwright B. J., Baker K., Mattick J. S. 'Touchdown' PCR to circumvent spurious priming during gene amplification. Nucleic Acids Res. 1991 Jul 25;19(14):4008–4008. doi: 10.1093/nar/19.14.4008. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Drexler H. G., Gignac S. M., Hu Z. B., Hopert A., Fleckenstein E., Voges M., Uphoff C. C. Treatment of mycoplasma contamination in a large panel of cell cultures. In Vitro Cell Dev Biol Anim. 1994 May;30A(5):344–347. doi: 10.1007/BF02631456. [DOI] [PubMed] [Google Scholar]
  5. Eremeeva M. E., Balayeva N. M., Raoult D. Purification of rickettsial cultures contaminated by mycoplasmas. Acta Virol. 1994 Aug;38(4):231–233. [PubMed] [Google Scholar]
  6. La Linn M., Bellett A. J., Parsons P. G., Suhrbier A. Complete removal of mycoplasma from viral preparations using solvent extraction. J Virol Methods. 1995 Mar;52(1-2):51–54. doi: 10.1016/0166-0934(94)00136-5. [DOI] [PubMed] [Google Scholar]
  7. McGarrity G. J., Vanaman V., Sarama J. Cytogenetic effects of mycoplasmal infection of cell cultures: a review. In Vitro. 1984 Jan;20(1):1–18. doi: 10.1007/BF02633326. [DOI] [PubMed] [Google Scholar]
  8. Messmer T. O., Black C. M., Thacker W. L. Mycoplasma contamination of chlamydiae isolated from clinical specimens. APMIS. 1994 Oct;102(10):793–796. doi: 10.1111/j.1699-0463.1994.tb05236.x. [DOI] [PubMed] [Google Scholar]
  9. Ossewaarde J. M., Rieffe M., Rozenberg-Arska M., Ossenkoppele P. M., Nawrocki R. P., van Loon A. M. Development and clinical evaluation of a polymerase chain reaction test for detection of Chlamydia trachomatis. J Clin Microbiol. 1992 Aug;30(8):2122–2128. doi: 10.1128/jcm.30.8.2122-2128.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Polak-Vogelzang A. A., Angulo A. F., Brugman J., Reijgers R. Survival of Mycoplasma hyorhinis in trypsin solutions. Biologicals. 1990 Apr;18(2):97–101. doi: 10.1016/1045-1056(90)90018-u. [DOI] [PubMed] [Google Scholar]
  11. Polak-Vogelzang A. A., Brugman J., Osterhaus A. D., Reygers R. Elimination of Mycoplasma from cell cultures by means of specific bovine antiserum. Zentralbl Bakteriol Mikrobiol Hyg A. 1987 Apr;264(1-2):84–92. doi: 10.1016/s0176-6724(87)80127-x. [DOI] [PubMed] [Google Scholar]
  12. Polak-Vogelzang A. A., Brugman J., Reijgers R. Comparison of two methods for detection of mollicutes (Mycoplasmatales and Acholeplasmatales) in cell cultures in the Netherlands. Antonie Van Leeuwenhoek. 1987;53(2):107–118. doi: 10.1007/BF00419507. [DOI] [PubMed] [Google Scholar]
  13. Ravaoarinoro M., Lecomte J. Evaluation of three methods for curing hybridomas from mycoplasma contamination. Hybridoma. 1988 Feb;7(1):79–86. doi: 10.1089/hyb.1988.7.79. [DOI] [PubMed] [Google Scholar]
  14. Rawadi G., Dussurget O. Advances in PCR-based detection of mycoplasmas contaminating cell cultures. PCR Methods Appl. 1995 Feb;4(4):199–208. doi: 10.1101/gr.4.4.199. [DOI] [PubMed] [Google Scholar]
  15. Roblin P. M., Dumornay W., Hammerschlag M. R. Use of HEp-2 cells for improved isolation and passage of Chlamydia pneumoniae. J Clin Microbiol. 1992 Aug;30(8):1968–1971. doi: 10.1128/jcm.30.8.1968-1971.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Rosendal S., Black F. T. Direct and indirect immunofluorescence of unfixed and fixed Mycoplasma colonies. Acta Pathol Microbiol Scand B Microbiol Immunol. 1972;80(4):615–622. doi: 10.1111/j.1699-0463.1972.tb00186.x. [DOI] [PubMed] [Google Scholar]
  17. Ursi J. P., Ursi D., Ieven M., Pattyn S. R. Utility of an internal control for the polymerase chain reaction. Application to detection of Mycoplasma pneumoniae in clinical specimens. APMIS. 1992 Jul;100(7):635–639. doi: 10.1111/j.1699-0463.1992.tb03978.x. [DOI] [PubMed] [Google Scholar]
  18. van Kuppeveld F. J., Johansson K. E., Galama J. M., Kissing J., Bölske G., van der Logt J. T., Melchers W. J. Detection of mycoplasma contamination in cell cultures by a mycoplasma group-specific PCR. Appl Environ Microbiol. 1994 Jan;60(1):149–152. doi: 10.1128/aem.60.1.149-152.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. van Kuppeveld F. J., van der Logt J. T., Angulo A. F., van Zoest M. J., Quint W. G., Niesters H. G., Galama J. M., Melchers W. J. Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl Environ Microbiol. 1992 Aug;58(8):2606–2615. doi: 10.1128/aem.58.8.2606-2615.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. van Kuppeveld F. J., van der Logt J. T., Angulo A. F., van Zoest M. J., Quint W. G., Niesters H. G., Galama J. M., Melchers W. J. Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification. Appl Environ Microbiol. 1993 Feb;59(2):655–655. doi: 10.1128/aem.59.2.655-.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES