TY - JOUR
T1 - Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles
AU - Rohr, Ulrich Peter
AU - Heyd, Florian
AU - Neukirchen, Judith
AU - Wulf, Marc Andre
AU - Queitsch, Iris
AU - Kroener-Lux, Gabriele
AU - Steidl, Ulrich
AU - Fenk, Roland
AU - Haas, Rainer
AU - Kronenwett, Ralf
N1 - Funding Information:
This work was supported by the Leukämie Liga e. V. Duesseldorf and Lions Club, Neuss.
PY - 2005/7
Y1 - 2005/7
N2 - 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.
AB - 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.
KW - Infectious assay
KW - qPCR
KW - rAAV-2
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U2 - 10.1016/j.jviromet.2005.03.006
DO - 10.1016/j.jviromet.2005.03.006
M3 - Article
C2 - 15893564
AN - SCOPUS:21044435643
SN - 0166-0934
VL - 127
SP - 40
EP - 45
JO - Journal of Virological Methods
JF - Journal of Virological Methods
IS - 1
ER -