Objective: To establish and verify a digital PCR method for the determination of recombinant adeno-associated virus type 5(rAAV5) genome titer, and to compare the performance of chip and droplet digital PCR methods. Methods: By examine the different lysing outcome from various concentrations of SDS or EDTA, the adeno-associated virus lysis method was optimized. To ensure other experimental conditions being consistent, the rAAV5 sample was amplified with WPRE primers in the droplet digital PCR method, and the annealing temperature was set to 60, 58, 54, 52 ℃. With amplification effect compared, the annealing temperature was optimized. Both chip and droplet digital PCR were tested to determine the genomic titer of rAAV5, and the consistency of the results of the two methods was compared. Results: The final concentration of 1% SDS and 62.5 mmol·L-1 EDTA was mixed 1∶1(v/v) and then rAAV5 was lysed. The measured genome copy number was high, the lysing outcome was good. At annealing temperature 54 ℃, the positive droplets in the droplet digital PCR and the separation effect of negative droplets was ideal, and the amplification specificity is the strongest. Comparing the two methods, it was found that the laboratory precision and recovery rates of the two determination results are similar, and the results of determining the same pyrolysis sample were relatively close (on the same order of magnitude). Conclusion: A preliminary digital PCR method has been established and validated, laying the foundation for further validation in the future.
ZHENG Hong-mei, QIN Xi, LI Yong-hong, YU Lei, BI Hua, RAO Chun-ming, ZHU Liu-qiang, YANG Jing-qing, SHI Xin-chang, ZHOU Yong
. Study on digital PCR methods for rAAV5 genomic titer determination*[J]. Chinese Journal of Pharmaceutical Analysis, 2023
, 43(11)
: 1826
-1832
.
DOI: 10.16155/j.0254-1793.2023.11.03
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