Quantitative Real-Time PCR for Rapid and Accurate Titration of Recombinant Baculovirus Particles
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This document describes a study that developed a quantitative real-time PCR (QPCR) method for rapidly and accurately determining baculovirus particle titers. The method uses Taqman probes and primers specific to baculovirus sequences. QPCR titers were found to be highly correlated with traditional plaque assay titration. The QPCR method offers faster and simpler titration than plaque assay or other methods, making it a valuable tool for optimizing baculovirus-based protein production.
Quantitative Real-Time PCR for Rapid and Accurate Titration of Recombinant Baculovirus Particles
- 1. Quantitative Real-Time PCR for Rapid and Accurate Titration of Recombinant Baculovirus Particles Bushra Hafeez
- 2. INTRODUCTION • The baculovirus expression system is widely used for the production of recombinant protein in insect cells. • Baculovirus can be amplified to very high titers in suspension culture facilitating the production of large quantities of recombinant protein. • In order to maximize and ensure reproducibility of protein production, it is important to obtain an accurate estimate of the recombinant virus titer.
- 3. INTRODUCTION • An improved QPCR titration method using Applied Biosystems Taqman fluorogenic probes is used. • It is faster and give increased accuracy across a wide range of virus titers. • This technology takes advantage of the 5’exonuclease activity of Taq polymerase, • To digest a probe.
- 4. INTRODUCTION • To hybridized between flanking PCR primers . • And labeled with two fluorescent dyes. • The measure of accuracy obtained using QPCR is verified by plaque assay, which is widely regarded as the most reliable method of baculovirus titration.
- 5. Baculovirus
- 7. Cell Culture and Recombinant Baculovirus Production • Sf9 insect cells are maintained at 27C in shakes flasks containing Sf900II media. • Recombinant baculovirus stocks are prepared using flashBAC. • Sf9 cells are removed from an exponentially growing culture and seeded in culture dishes. • Cells are transfected with lipofectin complexes of flashBAC DNA. • Transfer vector DNA and incubate at 27C.
- 8. Cont. … • Five days post-transfection, the culture medium containing recombinant virus is harvested. • 0.5 mL is used to inoculate shake flasks containing Sf9 cells and incubate at 27C on an orbital shaker. • Recombinant virus stocks are harvested 5 days post-infection. • clarified by centrifugation at low speed, and titrated by plaque assay in Sf21 cells.
- 9. Plaque Assay of Viral Titer • Cell culture dishes are seeded with Sf21 cells and allowed to settle for 1 h. • Viral stocks to be titrated are serially log diluted (1:10) inTC100 media supplemented with 10% fetal calf serum(FCS). • Virus inoculum is removed and agarose (2%) is overlaid onto the cells. • The cells are then stained with Neutral Red to visualize plaques.
- 10. Viral DNA extraction • Under sterile conditions, budded virus is removed from the viral stock to be titrated, and used for DNA extraction. • Viral DNA is extracted and purified using a High Pure Viral Nucleic Acid Kit. • Virus is lysed by addition of RNA- supplemented Binding Buffer and Proteinase K and incubated for 10 min at 72C.
- 11. Primers & Probes • Primer Express 3 software is used to design probes and primers. • The Taq Man probe is labeled with FAM (6- carboxy-fluorescein) at the 5’-end. • A quencher, TAMRA (6-carboxy- tetramethylrodamine) at the 3’-end. • Optimal concentrations of probe and primer are determined using a concentration matrix.
- 12. Quantitative PCR Conditions • QPCR reactions are prepared in 96-well optical plates using a CAS1200 liquid handler. • Each reaction contain Taqman probe, of each forward and reverse primer. • Quantitative PCR Super Mix-UDG and purified viral DNA. • DNA amplification is carried out using Real-Time PCR Sequence Detection System • Using cycling conditions of 50C UNG activation step.
- 13. Quantitative PCR Conditions • 95C enzyme activation. • 40 cycles of 95C denaturation. • 60C anneal/extension. • Duplicates of five 10-fold dilutions of the viral standard DNA viral DNA samples and no template controls are simultaneously subjected to analysis.
- 14. Statistical Analysis • Statistical analysis is carried out using triplicate cycle threshold (CT) values. • CT data is normalized by log10 transformation using SPSS. • Regression analysis is used to determine a QPCR-derived titer. • ANOVA is used to determine significant differences bw different groups of QPCR and plaque assay data.
- 16. Results and Discussion • A suitable target sequence is required that allow specific amplification of a single PCR amplicon. • Using the appropriate software, primers and probe are designed and their specificity is verified by gel electrophoresis. • A linear regression plot of CT values versus baculovirus titers is then produced.
- 17. Cont. … • CT value indicates the number of cycles taken to generate a fluorescent signal. • It is directly related to the template DNA concentration. • No significant difference is observed between titers produced by QPCR and plaque assay. • Confirming the validity of this technique as a rapid and accurate method of baculovirus titration.
- 19. Why QPCR is used? • Baculovirus (QPCR) titration method offers a much faster and simpler way to determine titers. • Based on the intensity of a fluorescent signal, generated during PCR amplification. • All other methods i.e., (plaque assay, EPD & antibody-based Assays) are • Labor intensive, • Time consuming, • Require a degree of expertise in cell culture and virus handling, • Sometimes producing results that can be difficult to interpret.