A Real-Time PCR Method to Rapidly Titer Adenovirus Stocks

  • Maria A. Thomas
  • Drew L. Lichtenstein
  • Peter Krajcsi
  • William S. M. Wold
Protocol
Part of the Methods in Molecular Medicine book series (MIMM, volume 130)

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 real-time 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.

Key Words

Adenovirus real-time PCR titration SYBR Green titer vector 

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Copyright information

© Humana Press Inc. 2007

Authors and Affiliations

  • Maria A. Thomas
    • 1
  • Drew L. Lichtenstein
    • 2
  • Peter Krajcsi
    • 3
  • William S. M. Wold
    • 1
  1. 1.Department of Molecular Microbiology and ImmunologySaint Louis University School of MedicineSt. Louis
  2. 2.VirRx, Inc.St. Louis
  3. 3.Research and Development DivisionSolvo BiotechnologyBudaorsHungary

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