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.%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.
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