A rapid Q-PCR titration protocol for adenovirus and helper-dependent adenovirus vectors that produces biologically relevant results.
A rapid Q-PCR titration protocol for adenovirus and helper-dependent adenovirus vectors that produces biologically relevant results.
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DOI：
10.1016/j.jviromet.2013.04.013
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发表时间：
2013-09
影响因子：
3.1
通讯作者：
Berk AJ
中科院分区：
文献类型：
--
作者：
Gallaher SD;Berk AJ
腺病毒用于细胞过程的研究和作为基因治疗中使用的表达载体。这些研究的成功和再现性部分取决于具有复制能力和辅助病毒依赖性腺病毒储备液的准确和有意义的滴度，这是有问题的，因为使用了各种不同的滴定方案。许多人使用物理滴定法来量化病毒颗粒的总数，但在估计活性方面很差。生物滴定方法，如噬斑测定，更具有生物学相关性，但耗时且不适用于辅助基因依赖性基因治疗载体。为了解决这个问题，开发了一种称为“感染性基因组滴定”的方案，其中在感染后约3小时从细胞核中分离病毒DNA，然后通过Q-PCR定量。该方法确保仅将生物活性病毒粒子计数作为滴度测定的一部分。该方法快速、稳健、灵敏、可重复，并且适用于所有形式的腺病毒。与其他基于Q-PCR的方法不同，通过该方案测定的滴度与生物活性良好相关。
Adenoviruses are employed in the study of cellular processes and as expression vectors used in gene therapy. The success and reproducibility of these studies is dependent in part on having accurate and meaningful titers of replication competent and helper-dependent adenovirus stocks, which is problematic due to the use of varied and divergent titration protocols. Physical titration methods, which quantify the total number of viral particles, are used by many, but are poor at estimating activity. Biological titration methods, such as plaque assays, are more biologically relevant, but are time consuming and not applicable to helper-dependent gene therapy vectors. To address this, a protocol was developed called “infectious genome titration” in which viral DNA is isolated from the nuclei of cells ~3 h post-infection, and then quantified by Q-PCR. This approach ensures that only biologically active virions are counted as part of the titer determination. This approach is rapid, robust, sensitive, reproducible, and applicable to all forms of adenovirus. Unlike other Q-PCR-based methods, titers determined by this protocol are well correlated with biological activity.
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影响因子：
64.5
作者：
CHOW, LT;GELINAS, RE;ROBERTS, RJ
通讯作者：
ROBERTS, RJ
影响因子：
5.4
作者：
Harui, A;Suzuki, S;Mitani, K
通讯作者：
Mitani, K
影响因子：
3.5
作者：
Crettaz, Julien;Olague, Cristina;Gonzalez-Aseguinolaza, Gloria
通讯作者：
Gonzalez-Aseguinolaza, Gloria
影响因子：
4.2
作者：
Kreppel, F;Biermann, V;Schiedner, G
通讯作者：
Schiedner, G
影响因子：
3
作者：
Ghosh, SS;Gopinath, P;Ramesh, A
通讯作者：
Ramesh, A