Objective: To explore the feasibility of multiplex PCR technique in recombinant adeno-associated virus(rAAV) genome integrity evaluation. Methods: A chip dPCR system was used to establish a dual PCR method targeting ITR region and gene of interest (or the 3’untranslated region) of rAAV genome. By proper dilution, a theoretical single copy distribution of template DNA was achieved in the PCR chip wells (the parentage of positive wells was less than 20%), the single positive wells represented the incomplete fragment, and the double positive wells represented the complete sequence between two sets of primer pairs. The integrity of rAAV genome was evaluated by testing the ratio of double positive wells. Different sample pretreatment methods (virus genomic DNA, virus particles, DNase Ⅰ treatment) were conducted to compare the test results. Results: The copy number measured by duplex PCR was basically consistent with that of single PCR, showing no obvious interference. Within a certain range, the measured copy number of samples was linearly correlated with the amount of templates, but the proportions of single positive and double positve was basically the same. Compared with untreated viral particles, the measured copy number of the extracted viral genome DNA was reduced, but the double positive rate was higher. The measured copy number of the virus samples treated with DNaseⅠ was significantly reduced, and the proportions of single positive and double positive were basically consistent with those of the untreated viral samples. Conclusion: Multiplex dPCR technology could be used to evaluate the genome integrity of rAAV products, showing a good application prospect.
YU Lei, WANG Guang-yu, FAN Wen-chao, SHI Xin-chang, ZHOU Yong
. Exploration and application of multiplex dPCR in rAAV genome integrity evaluation*[J]. Chinese Journal of Pharmaceutical Analysis, 2023
, 43(12)
: 1997
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DOI: 10.16155/j.0254-1793.2023.12.01
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