{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2023,12,19]],"date-time":"2023-12-19T05:24:45Z","timestamp":1702963485988},"reference-count":8,"publisher":"Cold Spring Harbor Laboratory","issue":"12","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cold Spring Harb Protoc"],"published-print":{"date-parts":[[2019,12]]},"abstract":"<jats:p>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 \u201cconcentration passage,\u201d 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<jats:sup>11<\/jats:sup> 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.<\/jats:p>","DOI":"10.1101\/pdb.prot095588","type":"journal-article","created":{"date-parts":[[2019,12,2]],"date-time":"2019-12-02T19:46:11Z","timestamp":1575315971000},"page":"pdb.prot095588","source":"Crossref","is-referenced-by-count":2,"title":["Detection Assay for Replication-Competent Adenovirus by Concentration Passage and Real-Time Quantitative Polymerase Chain Reaction (qPCR)"],"prefix":"10.1101","volume":"2019","author":[{"given":"Martin","family":"Lock","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mauricio","family":"Alvira","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"James","family":"Wilson","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Miguel","family":"Sena-Esteves","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Guangping","family":"Gao","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"246","published-online":{"date-parts":[[2019,12,2]]},"reference":[{"key":"2021111010173974000_2019.12.pdb.prot095588.1","doi-asserted-by":"publisher","DOI":"10.1089\/hum.1998.9.13-1909"},{"key":"2021111010173974000_2019.12.pdb.prot095588.2","doi-asserted-by":"crossref","first-page":"8459","DOI":"10.1128\/jvi.70.12.8459-8467.1996","article-title":"Molecular characterization of replication-competent variants of adenovirus vectors and genome modifications to prevent their occurrence","volume":"70","year":"1996","journal-title":"J Virol"},{"key":"2021111010173974000_2019.12.pdb.prot095588.3","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.prot095570"},{"key":"2021111010173974000_2019.12.pdb.prot095588.4","doi-asserted-by":"publisher","DOI":"10.1101\/pdb.prot095562"},{"key":"2021111010173974000_2019.12.pdb.prot095588.5","doi-asserted-by":"publisher","DOI":"10.1006\/viro.1997.8597"},{"key":"2021111010173974000_2019.12.pdb.prot095588.6","doi-asserted-by":"publisher","DOI":"10.1089\/10430340150218431"},{"key":"2021111010173974000_2019.12.pdb.prot095588.7","unstructured":"NIH Guidelines for Research Involving Recombinant DNA Molecules. (April) 2000."},{"key":"2021111010173974000_2019.12.pdb.prot095588.8","doi-asserted-by":"crossref","first-page":"89","DOI":"10.1016\/j.jviromet.2007.05.010","article-title":"A rapid and sensitive assay for detection of replication-competent adenoviruses by a combination of microcarrier cell culture and quantitative PCR","volume":"145","year":"2007","journal-title":"J Virol Methods"}],"container-title":["Cold Spring Harbor Protocols"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/syndication.highwire.org\/content\/doi\/10.1101\/pdb.prot095588","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,10]],"date-time":"2021-11-10T20:18:22Z","timestamp":1636575502000},"score":1,"resource":{"primary":{"URL":"http:\/\/www.cshprotocols.org\/lookup\/doi\/10.1101\/pdb.prot095588"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2019,12]]},"references-count":8,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2019,12,2]]},"published-print":{"date-parts":[[2019,12]]}},"alternative-id":["10.1101\/pdb.prot095588"],"URL":"https:\/\/doi.org\/10.1101\/pdb.prot095588","relation":{},"ISSN":["1940-3402","1559-6095"],"issn-type":[{"value":"1940-3402","type":"print"},{"value":"1559-6095","type":"electronic"}],"subject":[],"published":{"date-parts":[[2019,12]]}}}