Fast and reliable titration of recombinant adeno-associated virus type-2 using quantitative real-time PCR.
In this study, a quantitative real-time PCR (qPCR) was developed to determine genomic rAAV-2 titers using the Light-Cycler technology. Since the CMV promoter is the most commonly used promoter in gene therapeutic approaches, primers were designed which hybridize with the human CMV promoter sequence. PCR products were detected by the addition of SYBR green. qPCR of a 5 log spanning serial dilution of the vector plasmid containing one CMV promoter per plasmid molecule yielded a high amplification efficiency of 1.99 per cycle. To quantify the copy number of viral genomes, the qPCR curves of adeno-associated virus type 2 (AAV-2) samples were related to a standard curve assessed by the 5 log spanning serial vector plasmid dilution (0.01-100 pg DNA). For validation of the method, rAAV-2 preparations were analyzed by a standard method and qPCR in parallel. As standard method, flow cytometry was used for titration of infectious viral particles on HeLa cells using the Enhanced Green Fluorescent Protein as a marker. A significant correlation was found between the results obtained by flow cytometry and the results from the qPCR over a 5 log range (r=0.85, P<0.0001). The mean ratio between infectious rAAV-2 particles titrated via flow cytometry and genomic copies of rAAV-2 measured by qPCR of the same sample was 1:253. The higher titers found by qPCR might be due to multiple transduction of a single cell or to non-infectious particles generated during rAAV-2 preparation. In conclusion, qPCR is a fast and reliable method for determination of rAAV-2 titers and might be a powerful tool for standardization of rAAV-2 preparations particularly in the context of clinical studies.
D003587:Cytomegalovirus / Q000235:genetics; D004279:DNA, Viral / Q000032:analysis; D000229:Dependovirus / Q000235:genetics* / Q000502:physiology; D005434:Flow Cytometry; D005822:Genetic Vectors; D006367:HeLa Cells; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011995:Recombination, Genetic; D015203:Reproducibility of Results; D013997:Time Factors; D014771:Virion / Q000235:genetics / Q000502:physiology
D004279:DNA, Viral