A real-time PCR method to rapidly titer adenovirus stocks.

Maria A. Thomas, Drew L. Lichtenstein, Peter Krajcsi, William S.M. Wold

Research output: Contribution to journalArticle

16 Scopus citations

Abstract

A critical step in working with adenovirus (Ad) and its vectors is the accurate, reproducible, sensitive, and rapid measurement of the amount of virus present in a stock. Titration methods fall into one of two categories: determination of either the infectious or the particle (infectious plus noninfectious) titer. Determining the infectious titer of a virus stock by plaque assay has important limitations, including cell line-, researcher-, and laboratory-dependent variation in titer, and the length of time required to perform the assay (2-4 wk). A major drawback of particle titration methods is the lack of consistent correlation between the resultant titer and the infectious titer. To overcome these problems, a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay was developed that detects encapsidated full-length genomes. Importantly, there is a linear correlation between the titer determined by the realtime PCR assay and the infectious titer determined by a plaque assay. This chapter provides step-by-step guidance for preparing viral DNA, conducting the real-time PCR assay, and using the resultant data to calculate a viral titer.

Original languageEnglish
Pages (from-to)185-192
Number of pages8
JournalMethods in Molecular Medicine
Volume130
StatePublished - 2007

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    Thomas, M. A., Lichtenstein, D. L., Krajcsi, P., & Wold, W. S. M. (2007). A real-time PCR method to rapidly titer adenovirus stocks. Methods in Molecular Medicine, 130, 185-192.