Droplet digital PCR for growth kinetics of recombinant baculovirus expressing VP2 protein of porcine parvovirus

The utilization of insect cells for the baculovirus expression vector system to produce recombinant viral proteins and their virus-like particles (VLPs) is becoming popular. However, the instability of recombinant baculoviruses due to prolonged passaging and their lytic nature makes the harvest time a critical parameter for protein production. Droplet digital PCR (ddPCR) is an advanced technique for nucleic acid-based monitoring of kinetics of viruses and their related recombinant vectors. Its limited vulnerability to PCR inhibitors and irrelevance of any standard curve makes laboratories inclined towards the use of this assay. Objective: We applied ddPCR to determine the time at which maximum viral copies can be harvested based on our gene of interest i.e., VP2 of porcine parvovirus. We infected SF9 cells at low MOI and harvested the viral supernatant at different time intervals. A temperature gradient ddPCR was performed to optimize the annealing temperature. Results: ddPCR amplification revealed an increase in viral copy nos. 72 hr. post infection which peaked at 96 hr. post infection. These findings were comparable with the real-time PCR (qPCR) results, with ddPCR being 40-fold more sensitive in determining viral copy nos. The correlation between ddPCR andqPCR was found to be strong (r=0.8648, at 95% confidence interval). Conclusion: These results indicate that ddPCR assay has high sensitivity and in comparison, to real-time PCR has a lower turnaround time, although instruments/reagent availability may impact the choice of assay. We conclude that ddPCR assay has the potential for quantitative analysis of virus infection.