Identification of recombination baculovirus and determination of virus titer with fluorescence quantitative PCR assay
- VernacularTitle:荧光定量PCR用于重组杆状病毒鉴定及病毒滴度检测的研究
- Author:
Bo SHEN
;
Zhefeng MENG
;
Ying PENG
;
Jianxin L
- Publication Type:Journal Article
- Keywords:
Fluorescence quantitative PCR;
Identification of recombinant baculovirus;
Virus titer
- From:
Chinese Journal of Immunology
2000;0(11):-
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To develop a real-time PCR assays based on TaqMan chemistry 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 series diluted primary viral stocks were used for plaque assay and DNA extraction.Bacmid(baculovirus plasmid) was 10-fold series 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(101 to 108 copies) from 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.
