Details

Autor(en) / Beteiligte

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

Titel

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

Ist Teil von

• Journal of virological methods, 2005-07, Vol.127 (1), p.40-45

Ort / Verlag

London: Elsevier B.V

Erscheinungsjahr

2005

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.