Fluctuations in AAV genome quantification via digital PCR affected by heat-based virion lysis

Adeno-associated virus (AAV) vectors are central to gene therapy, making precise genome quantification essential for product quality and dose determination. We systematically assessed digital PCR (dPCR) on the QIAcuity platform to quantify recombinant AAV2 and AAV8 genomes, examining how assay design, amplicon length, and heat-based sample preparation affect results. Across multiple genomic targets, shorter amplicons consistently yielded higher copy numbers than longer ones leading to a quantification deviation of up to ∼48%. The results point to heat-induced genomic fragmentation as the main cause of the observed effect. These findings highlight that dPCR-based AAV quantification is highly sensitive to amplicon length and sample preparation. We propose the use of the employed multitarget assay set to evaluate AAV genome extraction procedures, the quality of AAV genomic DNA extracts, and ultimately the AAV genome integrity.