Quantitative real-time PCR for titration of infectious recombinant AAV-2 particles.
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.
Rohr
Ulrich-Peter
UP
Klinik für Haematologie, Onkologie und klinische Immunologie, Heinrich-Heine-Universitaet Duesseldorf, Moorenstrasse 5, D-40225 Duesseldorf, Germany. urohr-uni-duesseldorf@gmx.de
Heyd
Florian
F
Neukirchen
Judith
J
Wulf
Marc-Andre
MA
Queitsch
Iris
I
Kroener-Lux
Gabriele
G
Steidl
Ulrich
U
Fenk
Roland
R
Haas
Rainer
R
Kronenwett
Ralf
R
D002460:Cell Line; D004277:DNA, Single-Stranded / Q000032:analysis / Q000378:metabolism; D004279:DNA, Viral / Q000032:analysis / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D005822:Genetic Vectors / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D015203:Reproducibility of Results; D015719:Single-Strand Specific DNA and RNA Endonucleases; D014779:Virus Replication
D004277:DNA, Single-Stranded; D004279:DNA, Viral; D015719:Single-Strand Specific DNA and RNA Endonucleases