Characterization of high-capacity adenovirus production by the quantitative real-time polymerase chain reaction: a comparative study of different titration methods
Characterization of high-capacity adenovirus production by the quantitative real-time polymerase chain reaction: a comparative study of different titration methods
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DOI：
10.1002/jgm.1236
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发表时间：
2008-10-01
影响因子：
3.5
通讯作者：
Gonzalez-Aseguinolaza, Gloria
中科院分区：
文献类型：
--
作者：
Crettaz, Julien;Olague, Cristina;Gonzalez-Aseguinolaza, Gloria
背景高容量腺病毒(HC-Ad)在多种疾病的治疗中具有广阔的应用前景。这种载体临床应用的主要缺点是难以大规模生产，以及缺乏标准化的生产和滴定方法。在本研究中，我们比较了文献中用于鉴定HC-Ad产物的不同方法。方法获得两种携带小鼠IL-12基因的HC-Ad产物。用不同的方法测定病毒滴度、腺病毒辅助病毒污染以及病毒颗粒浓度：(I)用分光光度法和空斑试验定量病毒总颗粒以估计第一代(FG)辅助病毒的污染；(Ii)直接从病毒库中定量HC-Ad和FG辅助病毒基因组；(Iii)用狭缝杂交技术定量细胞感染后的病毒基因组；(Iv)用qPCR方法定量病毒滴度。第一种方法高估了HC-Ad的滴度，低估了FG-Helper-Ad的污染度，没有得到关于HC-Ad感染性的信息。病毒群的定量聚合酶链式反应分析更敏感和准确，但关于传染性的信息仍然未知，FG-Helper-Ad污染被高估。用狭缝印迹DNA杂交法和定量聚合酶链式反应(QPCR)方法定量检测HC-Ad和FG-helper-Ad感染单位具有同样的敏感性和准确性。结论本研究结果为HC-Ad的临床应用提供了一种标准化方法。定量检测HC-Ad是一种快速、安全、可靠的检测HC-Ad和FG-helper-Ad颗粒及感染单位的方法。版权所有(C)2008 John Wiley&Sons，Ltd.
Background High-capacity adenoviruses (HC-Ad) hold great promise for the treatment of many diseases. The major drawbacks for the clinical application of this vector concern difficulties with respect to large-scale production, and the absence of standardized methods for production and titration. in the present study, we compare the different methods found in the literature for characterizing HC-Ad production.Methods Two productions of the HC-Ad carrying murine IL-12 gene were obtained. The viral titer and adenovirus-helper contamination as well Lis viral particle concentration of both productions were determined using different methods: (i) quantification of total viral particles by spectrophotometry and plaque assay to estimate first-generation (FG)-helper-Ad contamination; (ii) quantification of HC-Ad and FG-helper-Ad genomes by the quantitative polymerase chain reaction (qPCR) directly from viral stock, (iii) quantification of viral genomes after cell infection by the slot-blot hybridization assay and (iv) qPCR.Results Dramatic differences with respect to viral titer were found depending on the method used. The first method overestimates HC-Ad titer and underestimates FG-helper-Ad contamination and no information on the infectivity of the HC-Ad is obtained. qPCR analysis of viral stock is more sensitive and accurate, but information about infectivity remains unknown and FG-helper-Ad contamination is overestimated. Quantification of HC-Ad and FG-helper-Ad infectious units by-slot blot DNA hybridization and qPCR assay are found to be equally sensitive and accurate.Conclusions The results of the present study demonstrate that a standardized method should be developed for HC-Ad characterization for future clinical applications of this vector. Quantification of HC-Ad production by qPCR is a fast, safe and reliable method for determining HC-Ad and FG-helper-Ad particles and infectious units. Copyright (C) 2008 John Wiley & Sons, Ltd.