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A real time quantitative PCR-based method for the detection and quantification of simian virus 40.
L Shi1, J Ho, L A Norling
1Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Summary
A new real-time quantitative PCR method accurately detects and quantifies simian virus 40 (SV40) DNA. This sensitive assay offers reliable viral detection for research and product development.
Area of Science:
- Molecular Biology
- Virology
- Biotechnology
Background:
- Simian virus 40 (SV40) detection is crucial for pathogenic investigations and biological product safety.
- Existing methods for SV40 detection and quantification have limitations in accuracy, precision, and scope.
Purpose of the Study:
- To develop and validate a real-time quantitative PCR (qPCR) method for SV40 detection and quantification.
- To establish a sensitive, accurate, and reproducible assay for SV40 DNA analysis.
Main Methods:
- Utilized Taq DNA polymerase's 5' to 3'-exonuclease activity with a dual-labelled fluorogenic probe.
- Employed the PRISM 7700 sequence detection system for real-time PCR product monitoring.
- Developed an efficient sample preparation procedure for consistent SV40 DNA recovery.
Main Results:
- Achieved accurate, precise, and reproducible quantification of SV40 DNA over a 100,000-fold dynamic range.
- Established a minimum detection level of 6.4 copy equivalents/µL of SV40 viral particles.
- Demonstrated robustness against high concentrations of protein and cellular DNA in samples.
Conclusions:
- The developed real-time qPCR method offers significant advantages over existing PCR and cell-based assays for SV40 detection.
- This assay facilitates SV40 pathogenic investigation and viral clearance evaluations for biological product development.