Identification of recombinant baculovirus and determination of virus titer with fluorescence quantitative PCR assay

AIM: To develop a real-time PCR assay based on TaqMan technology for the identification of recombinant baculovirus and determination of virus physical titers in Bac-to-Bac system.METHODS: The recombinant baculovirus containing human IL-18 gene was produced using Bac-to-Bac system.A 10-fold serially diluted primary viral stock was used for plaque assay and DNA extraction.Bacmid (baculovirus plasmid) was 10-fold serially diluted and served as standards.Real-time PCR amplification of the IL-18 gene was performed in triplicate for each diluted recombinant virus.At the same time,plaque assays were performed using overlay agarose method.RESULTS: The standard linear range (101 to 108 copies) for quantitation was achieved with the standard curve.We also find that the“vg/mL”titer value is generally about 10 times than“pfu/mL”titer of the same recombinant virus stock.CONCLUSION: A TaqMan real-time PCR method is established to identify the recombinant baculovirus and determine the“vg/mL”titer of virus.The method is rapid and quantitative over a wide range of virus titers.