Establishment and verification of a digital PCR detection method for genome titer of adeno-associated virus gene therapy products
Objective To establish and preliminarily verify a digital polymerase chain reaction(dPCR)method for the detection of genome titer of adeno-associated virus(AAV)gene therapy products.Methods Through dPCR detection of the same batch of AAV gene therapy products,the primers,probes,sample treatment methods and concentrations of primer and probe were optimized.The specificity,linearity,relative accuracy and repeatability of the optimized method were veri-fied,and the limit of quantitation(LOQ)and detection range were determined.Results NFS-05 vector titer 5.0 crop primer probe was selected,the final primer concentration was 800 nmol/L,the final probe concentration was 400 nmol/L,and the sample pretreatment method was digestion by Proteinase K.The established method had good specificity.The correlation coefficient(R2)of the standard curve was greater than 0.999 in the range of 3.3 x 105-1.3 x 108 vg/mL.The relative accuracy was between 70％and 130％,the CV of repeatability was not more than 15％,and the LOQ was 3.3 x 105 vg/mL.Conclusion The established dPCR method has good specificity,linearity,accuracy and repeatability,and can be applied to the determination of the genome titer of NFS-05 recombinant adeno-associated virus(rAAV)stock solution products.
Digital polymerase chain reaction(dPCR)Adeno-associated virus(AAV)Genome titerGene therapy
NETL
NSTL
万方数据
