实时聚合酶链反应
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聚合酶链反应
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血清型
载体(分子生物学)
病毒学
分子生物学
腺病毒科
HEK 293细胞
聚合酶
生物
遗传增强
遗传学
基因
重组DNA
作者
Martin Lock,Mauricio R. Alvira,James M. Wilson,Miguel Sena‐Esteves,Guangping Gao
出处
期刊:CSH Protocols
[Cold Spring Harbor Laboratory]
日期:2019-12-01
卷期号:2019 (12): pdb.prot095588-pdb.prot095588
被引量:2
标识
DOI:10.1101/pdb.prot095588
摘要
The sensitivity of an assay for replication-competent adenoviruses (RCAs) can often be enhanced by biological amplification of the RCAs with serial passage. Here, we describe an extension of this technique, termed “concentration passage,” in which RCA replicated during the first plating of the vector is collected and concentrated onto one-tenth of the original number of cells. This significantly increases the chances of detecting the RCAs. Combining this approach with the use of quantitative polymerase chain reaction (qPCR) for sensitive detection of the RCA E1 gene, we are able to reach levels of sensitivity of 1 IU of RCAs in 10 11 vector particles. The protocol described here is tailored for HuAd5 vectors using wild-type HuAd5 as the RCA surrogate. However, we have also adapted this technique with similar sensitivity to vectors based on other adenovirus serotypes. If other adenovirus serotypes are assayed, careful consideration should be given to the appropriate RCA surrogate. Strictly speaking, if the vector is propagated in HEK-293 or similar cell lines, the RCA surrogate should be a hybrid virus containing the HuAd5 E1 gene.
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