Rapid assessment of AAV genome integrity using multiplexed PCR with single-molecule detection

Adeno-associated virus (AAV) genome integrity is a critical quality attribute that directly impacts therapeutic efficacy. During AAV production, multiple forms of incomplete or aberrant genomic impurities can arise, including partially packaged genomes, snap-back genomes, and residual genomic impurities derived from producer plasmids. Traditional methods for assessing genome integrity, such as DNA electrophoresis–based assays, provide limited resolution, while more advanced approaches, such as long-read sequencing platforms, require extensive sample preparation and analysis time. There is an unmet need for rapid, quantitative methods to assess AAV genome integrity across diverse sample types and production scales. 

Single-molecule PCR with universal multiplexing provides a rapid, quantitative method for assessing AAV genome integrity that distinguishes between partial and intact viral genomes. The ability to simultaneously measure genome integrity in a single multiplex reaction with a high dynamic range offers significant workflow advantages for AAV production and quality control.