Quantification of Adeno-Associated Viral Genomes in Purified Vector Samples by Digital Droplet Polymerase Chain Reaction

dd_PCR is a widely used technique for determining the AAV genome titer, but a consensus protocol is currently lacking. Our protocol is a validated step-by-step guide on how to prepare samples and perform dd_PCR for accurate genome titration. Compared with qPCR, dd_PCR offers several advantages, including increased precision, greater robustness, and a more absolute and direct quantification of target sequences without the need for standard curves.

Furthermore, with a well-designed primer probe set, dd_PCR allows for specific tragedy in detection as opposed to other techniques that detect all DNA. Developing a standardized consensus protocol for vector genome quantification will improve reliability and uniformity in recombinant AAV quality control across different laboratories. This will facilitate both the research and clinical applications recombinant AAV vectors for the wider community.

To begin, vortex the adeno-associated virus or AAV sample and centrifuge to ensure all liquid remains at the bottom of the tube. Add 45 microliters of DNase I containing solution to a 0.2 milliliter PCR 8-tube strip tube. Transfer five microliters of the AAV sample into the tube containing the DNA solution.

After vortexing, centrifuge the sample briefly to ensure all liquid remains at the bottom. Using a thermocycler, incubate the samples for one hour at 37 degrees Celsius, then cool them down to four degrees Celsius. Next, prepare appropriate dilution of the DNA's treated sample in AAV dilution buffer in a fresh 0.2 milliliter PCR 8-tube strip.

After serial dilution, vortex the samples and centrifuge to ensure all the liquid remains at the bottom. Combine the forward and reverse primers, probe dd_PCR super mix and DNase-free water in the required volumes. Vortex the master mix and briefly centrifuge the tube.

Then transfer 19.8 microliters of the master mix into each tube on a fresh 0.2 milliliter PCR 8-tube strip. To begin, prepare DNase I treated AAV sample and the dd_PCR master mix. Add 2.2 microliters of the diluted sample to 19.8 microliters of dd_PCR master mix in each tube of the PCR 8-tube strip and mix well.

Transfer 20 microliters of the mixture into the wells contained in the middle sample row of a droplet-generating cartridge. Then transfer 60 microliters of droplet generation oil to the wells contained in the lower oil row of the droplet-generating cartridge. Place a rubber gasket over the droplet-generating cartridge, and then place it in the droplet generator.

After the droplets are generated, use an eight-channel pipette to slowly transfer 42.5 microliters of solution from the droplet generation cartridge to a multi-well PCR plate. Seal the PCR plate with an aluminum foil cover using a heat ceiling machine for five seconds at 180 degrees Celsius. For amplification, place the plate in a thermal cycler with a 96-deep well reaction module and close it securely and run the PCR program.

Once done, load the plate into a droplet reader, input the required information into the system software and start the reading. A clear separation between positive and negative droplets was observed in the 1D amplitude plot, suggesting a successful measurement. The second 1D amplitude plot showed droplet rain with droplets scattered between the positive and negative clouds, indicating a potential issue with measurement accuracy.

The output data from an AAV run showed consistent results when one sample was measured in duplicates at two different dilutions.