, Volume 38, Issue 1–3, pp 147–153 | Cite as

Real-time RT-PCR detection of retroviral contaminations of cells and cell lines

  • Katja Müller
  • Manfred Wirth


We have developed a fast and sensitive on-line detection method for retroviruses using the PCR technology. The assay utilizes the endogenous reverse transcriptase activity in retroviral particles. In the presence of active reverse transcriptase, bacteriophage MS2 RNA is transcribed into cDNA and is subsequently amplified in a SYBR-Green-type LightCycler™ reaction. The method allows a qualitative and quantitative monitoring of RT-activity, is several orders of magnitude more sensitive than a standard RT assay and has a time requirement of 2.5 hours from harvest to result. The methodis useful for monitoring of cells and cell-derived products, viral vectors and recombinant proteins for the presence ofreplication-competent retroviruses (RCRs).

adventitious agents on-line RT-PCR quantitation retrovirus viral diagnosis virus 


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  1. Anderson KB (1983) Entry of murine retroviruses into mouse fibroblasts. Virology 125: 85-98.Google Scholar
  2. Arnold BA, Heppler RW& Keller PM (1998) One-step fluorescent probe product-enhanced reverse transcriptase assay. Biotechniques 25: 98-106.Google Scholar
  3. Chang A, Ostrove JM& Bird RE (1997) Development of an improved product enhanced reverse transcriptase assay. J Virol Methods 65: 45-54.Google Scholar
  4. Cosset F-L, Takeuchi Y, Battini J-L, Weiss RA& Collins MKL (1995) High-Titer packaging cells producing recombinant retro-viruses resistant to human serum. J Virol 69: 7430-7436.Google Scholar
  5. Deo Y, Ghebremariam H& Cloyd M (1994) Detection and characterization of murine ecotropic recombinant virus in myeloma and hybridoma cells. Hybridoma 13: 69-76.Google Scholar
  6. De Wit C, Fautz C& Xu Y (2002) Real-time quantitative PCR for retrovirus-like particle quantification in CHO cell culture. Biologicals 28: 137-148.Google Scholar
  7. Froud SJ, Birch J, MacLean C, Shepher AJ& Smith KT (1997) Animal Cell Technology: Products of Today, Prospects for Tomorrow Viral Contaminants Found in Mouse Cell Lines used in the Production of Biological Products (pp. 681-686) Kluwer Academic Publishers, Dordrecht.Google Scholar
  8. Goff S, Traktman P& Baltimore D (1981) Isolation and properties of Moloney murine leukemia virus mutants use of a rapid assay for release fo virion reverse transcriptase. J Virol 38: 239-248.Google Scholar
  9. Heinemeyer T, Klingenhoff A, Hansen W, Schumacher L, Hauser H& Wirth M (1997) A sensitive method for the detection of murine C-Type retroviruses. J Virol Methods 63: 155-165.Google Scholar
  10. Karreman C, Karreman S& Hauser H (1996) Retroviral infection of syrian hamster BHK cells depends on age and susceptibility toward sialidase. Virology 220: 46-50.Google Scholar
  11. Lovatt A, Black J, Galbraith D, Doherty I, Moran MW, Shepherd AJ, Griffen A, Bailey A, Wilson N& Smith KT (1999) High througput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse transcriptase assay and its comparison to conventional detection methods. J Virol Methods 82: 185-200.Google Scholar
  12. Lugert R, König H, Kurth R& Tönjes RR (1996) Specific suppression of false-positive signals in the product-enhanced reverse transcriptase assay. BioTechniques 20: 210-217.Google Scholar
  13. Markowitz D, Goff S& Bank A (1988) A safe packaging line for gene transfer: Separating viral genes on two different plasmids. J Virol 62: 1120-1124.Google Scholar
  14. Maudru T& Peden K (1997) Elimination of background signals in a modified polymerase chain reaction-based reverse transcriptase assay. J Virol Methods 66: 247-261.Google Scholar
  15. Miller AD& Buttimore C (1986) Redesign of retrovirus packaging cell lines to avoid recombination leading to helper virus production. Mol Cell Biol 6: 2895-2902.Google Scholar
  16. Morgenstern JP& Land H (1990) Advanced mammalian gene transfer: High titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line. Nucleic Acids Res 18: 3587-3596.Google Scholar
  17. Pyra H, Böni J& Schupbach J (1994) Ultrasensitive retrovirus detection by a reverse transcriptase assay based on product enhancement. Proc Natl Acad Sci USA 91: 1544-1548.Google Scholar
  18. Silver J, Maudru T, Fujita K& Repaske R (1993) A RT-PCR assay for the enzyme activity of reverse transcriptase capable of detecting single virions. Nucleic Acids Res 21: 3593-3594.Google Scholar
  19. Towers GJ, Stockholm D, Labbrousse-Najburg V, Carlier F, Danos O& Pages J-C (1999) One step screening of retroviral producer clones by real time quantitative PCR. J Gene Med 1: 352-359.Google Scholar

Copyright information

© Kluwer Academic Publishers 2002

Authors and Affiliations

  • Katja Müller
    • 1
  • Manfred Wirth
    • 1
  1. 1.Molecular BiotechnologyGerman Research Centre of Biotechnology, GBFBraunschweigGermany

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