Identification of recombinant baculovirus and determination of virus titer with fluorescence quantitative PCR assay / 中国病理生理杂志

ABSTRACT

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.