A real-time PCR assay for quantification of canine adenoviral vectors.
The pre-existing humoral and cellular immunity found in the great majority of the population raises concerns about the clinical efficacy and safety of vectors derived from ubiquitous human adenovirus serotypes. To alleviate these concerns, canine adenovirus type 2 vectors (CAV-2) were developed. Owing to their extraordinary neuronal tropism and efficient axonal retrograde transport, CAV-2 vectors hold great promise for the treatment of neurodegenerative diseases. The development and validation of a SYBR Green qPCR assay for determination of CAV-2 titers is reported in the present study. This method uses specific primers designed to amplify a small genomic fragment of CAV-2 structural genes (pVI-hexon). The method was accurate and reproducible as determined by the low intra- and inter-assay variability (<15% R.S.E.). It is sensitive and useful over a 5-log range (1 x 10(3) to 1 x 10(7)genome copies/reaction). The assay can be used to quantify purified vector samples as well as crude viral lysates. The titers obtained by qPCR correlated well with both, those obtained by OD(260) and TCID(50) as indicated by the high coefficients of determination obtained by regression analysis (r(2)>0.83). The development of this simple and rapid CAV-2 quantitation method should be helpful for process development and monitoring.
Segura
María Mercedes
MM
Department of Biochemistry and Molecular Biology, Center of Animal Biotechnology and Gene Therapy (CBATEG), Universitat Autònoma de Barcelona, Barcelona 08193, Spain.
Monfar
Mercè
M
Puig
Meritxell
M
Mennechet
Franck
F
Ibanes
Sandy
S
Chillón
Miguel
M
D006516:Adenoviruses, Canine / Q000235:genetics / Q000302:isolation & purification*; D000818:Animals; D002460:Cell Line; D004279:DNA, Viral / Q000032:analysis*; D004285:Dogs; D005822:Genetic Vectors / Q000032:analysis*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods*
D004279:DNA, Viral