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

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E.coli菌株DH10 Bac中构建重组杆状病毒穿梭质粒（Bacmid）和在昆虫细胞中构建含人IL-18基因的重组杆状病毒,纯化的重组Bacmid作为PCR检测的标准模板,由昆虫细胞中收获的病毒母液用于空斑测定和病毒DNA提取.以10倍梯度稀释的重组Bacmid作为标准模板,进行荧光定量PCR扩增IL18基因片段并绘制标准曲线,然后以提取的重组杆状病毒DNA作为模板,采用同样体系进行实时PCR反应检测.同时,以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,并在较大的线性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.

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

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.