摘要
目的:建立一种高效?简便的荧光实时定量PCR方法,用于重组杆状病毒鉴定及病毒滴度的检测。方法:利用Bac-to-Bac载体系统在昆虫细胞中构建含人IL-18基因的重组杆状病毒,收获的病毒母液以10倍梯度系列稀释后,提取病毒基因组DNA。以10倍梯度稀释的重组杆状病毒穿梭质粒(bacmid)作为标准模板,进行荧光定量PCR反应扩增IL-18基因片段并绘制标准曲线,然后以上述的重组杆状病毒基因组DNA作为模板,采用同样体系进行实时PCR反应检测,同时用琼脂糖空斑法测定病毒母液的滴度。结果:成功构建了重组杆状病毒并建立了病毒滴度的实时荧光PCR检测方法。运用标准模板进行的PCR反应显示该方法的线形范围为101-108拷贝,病毒母液的DNA拷贝浓度(vg/mL)值约为空斑检测的滴度pfu/mL值的10倍。结论:荧光定量PCR方法可灵敏快速地鉴定重组杆状病毒,并在较大的线形范围内检测重组杆状病毒滴度,较之空斑法更准确地反映了重组杆状病毒的实际数量。
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 （10^1 to 10^8 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 methed is rapid and quantitative over a wide range of virus titers.
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2007年第8期1623-1626,共4页
Chinese Journal of Pathophysiology
关键词
荧光定量PCR
重组病毒鉴定
病毒滴度
Fluorescence quantitative PCR
Identification of recombinant baculovirus
Virus titer
