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A sensitive method for the detection of murine C-type retroviruses
T Heinemeyer1, A Klingenhoff, W Hansen
1Department of Regulation and Differentiation, Gesellschaft für Biotechnologische Forschung, Braunschweig, Germany.
Journal of Virological Methods
|January 1, 1997
Summary
This study presents a highly sensitive RT-PCR assay for detecting mouse retroviruses. The method accurately identifies exogenous and endogenous murine C-type retroviruses in cell cultures, crucial for biopharmaceutical production.
Area of Science:
- Virology
- Molecular Biology
- Biotechnology
Background:
- Murine C-type retroviruses can contaminate cell cultures, impacting research and biopharmaceutical production.
- Distinguishing between exogenous and endogenous retroviruses is critical for accurate diagnostics.
Purpose of the Study:
- To develop a sensitive and specific assay for detecting murine C-type retroviruses.
- To differentiate between exogenous and endogenous retroviral infections in cell cultures.
Main Methods:
- Development of a nested degenerate primer set for RT-PCR.
- Application of DNAse I pretreatment and heat inactivation for sample preparation.
- Ultrasensitive detection of Moloney murine leukemia virus (MoMuLV) proviral DNA and infectious retroviruses.
Main Results:
- The assay detected 100 attograms of MoMuLV proviral DNA.
- It identified 1-10 infectious mouse C-type retroviruses per 10 microliters of supernatant.
- High specificity differentiated mouse retroviruses from other types and species.
Conclusions:
- The developed RT-PCR assay is a valuable tool for screening animal cell cultures for mouse retroviral contamination.
- This method aids in ensuring the safety and integrity of cell lines used for producing foreign proteins, such as hybridomas.