Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles

Ulrich Peter Rohr, Florian Heyd, Judith Neukirchen, Marc Andre Wulf, Iris Queitsch, Gabriele Kroener-Lux, Ulrich G. Steidl, Roland Fenk, Rainer Haas, Ralf Kronenwett

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

In this report, we present a fast, reliable and easy to perform method to quantify infectious titers of recombinant AAV-2 (rAAV-2) particles using the LightCycler technology, which is independent from the therapeutic transgene and without the presence of a marker gene. The method is based on the life cycle of AAV-2: after infection of the host cell, the single stranded (ss) AAV-2 genome is converted into a double stranded (ds) form. Following infection with rAAV-2, HeLa cells were lysed and ssDNA of transcriptionally inactive particles were efficiently removed by ssDNA-specific S1 nuclease digestion. The remaining viral dsDNA can be quantified by quantitative real-time PCR (qPCR). For validation of the new method, rAAV-2 preparations were analyzed by two other standard methods for titration of infectious particles in parallel, i.e. the infectious center assay (ICA) as well as flow cytometry using GFP as a marker. Comparing the infectious titers of 40 different AAV-2 fractions assessed by qPCR with the titers determined by FACS analysis a significant correlation (r = 0.87, p < 0.001) with a mean ratio of the titers assessed by qPCR and FACS of 1.92 (S.D. ± 1.59) was found. Further, the titers of seven rAAV-2 fractions using qPCR and ICA covering 5 log ranges were compared and a significant correlation was found between the results (r = 0.80, p < 0.001) with a mean ratio of 3.38 (S.D. ± 1.79), respectively.

Original languageEnglish (US)
Pages (from-to)40-45
Number of pages6
JournalJournal of Virological Methods
Volume127
Issue number1
DOIs
StatePublished - Jul 2005
Externally publishedYes

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Real-Time Polymerase Chain Reaction
Infection
Life Cycle Stages
Transgenes
HeLa Cells
Digestion
Flow Cytometry
Genome
Technology
Genes
Therapeutics

Keywords

  • Infectious assay
  • qPCR
  • rAAV-2

ASJC Scopus subject areas

  • Virology

Cite this

Rohr, U. P., Heyd, F., Neukirchen, J., Wulf, M. A., Queitsch, I., Kroener-Lux, G., ... Kronenwett, R. (2005). Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles. Journal of Virological Methods, 127(1), 40-45. https://doi.org/10.1016/j.jviromet.2005.03.006

Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles. / Rohr, Ulrich Peter; Heyd, Florian; Neukirchen, Judith; Wulf, Marc Andre; Queitsch, Iris; Kroener-Lux, Gabriele; Steidl, Ulrich G.; Fenk, Roland; Haas, Rainer; Kronenwett, Ralf.

In: Journal of Virological Methods, Vol. 127, No. 1, 07.2005, p. 40-45.

Research output: Contribution to journalArticle

Rohr, UP, Heyd, F, Neukirchen, J, Wulf, MA, Queitsch, I, Kroener-Lux, G, Steidl, UG, Fenk, R, Haas, R & Kronenwett, R 2005, 'Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles', Journal of Virological Methods, vol. 127, no. 1, pp. 40-45. https://doi.org/10.1016/j.jviromet.2005.03.006
Rohr, Ulrich Peter ; Heyd, Florian ; Neukirchen, Judith ; Wulf, Marc Andre ; Queitsch, Iris ; Kroener-Lux, Gabriele ; Steidl, Ulrich G. ; Fenk, Roland ; Haas, Rainer ; Kronenwett, Ralf. / Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles. In: Journal of Virological Methods. 2005 ; Vol. 127, No. 1. pp. 40-45.
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