Serotype-specific detection of adeno-associated virus during laboratory preparation.
Adeno-associated virus (AAV) is a small single-stranded DNA member of the family Parvoviradae with at least eight recognized human serotypes, several of which are being studied as candidate vectors for gene therapy. When multiple serotypes are handled in the same laboratory, it is critical to know the serotype of a sample with certainty. Here, a rapid and reliable PCR-based method is presented for the identification of serotypes 2, 3B or 6 and for screening for cross contamination. The PCR assay is based on the use of differentially annealing, serotype-specific primer pairs targeted to the hypervariable regions of the capsid coding region of the AAV genome. Identity is determined by the presence of a PCR product of size specific to each serotype. Multiplexing the reaction allows all serotypes to be queried in a single reaction tube and greatly diminishes the presence of false positives. The method is capable of detecting as little as 25 fg of a contaminating serotype and is potentially extensible to other AAV serotypes.
Mitchell
Daniel A J
DA
Institute of Molecular Biophysics, Kasha Laboratory of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-4381, USA.
O'Donnell
Jason
J
Hare
Joan T
JT
Chapman
Michael S
MS
D001483:Base Sequence; D036022:Capsid Proteins / Q000235:genetics; D004279:DNA, Viral / Q000235:genetics*; D000229:Dependovirus / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*; D004587:Electrophoresis, Agar Gel; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012703:Serotyping
D036022:Capsid Proteins; D004279:DNA, Viral