Quantification of Adeno-Associated Viral Vectors by Digital Droplet PCR

           Quantification of Adeno-Associated Viral Vectors by Digital Droplet PCR

Droplet digital PCR (ddPCR) for AAV Quantitation

Droplet digital PCR (ddPCR) is a new PCR technique that directly quantifies DNA copies with an unparalleled degree of precision and without the need for a standard curve or for a high degree of amplification efficiency. These properties allow accurate quantification of both single-stranded and self-complementary AAV genomes. ddPCR method has demonstrated superior sensitivity on the absolute quantification of single-stranded AAV vector genomes by ddPCR over a standard qPCR assay. 

ddPCR is suitable when 

• qPCR is sensitive to the presence of inhibitors in the PCR reaction 
• qPCR shows erroneous results with the secondary structure of DNA templates 
• qPCR is dependent on a DNA standard curve for quantification. 

Protocol for ddPCR 

Preparation of AAV Genomes for ddPCR

The preparation of genomic DNA from viral vectors depends on the source of DNA. The purification of the AAV genomes from the manufacturing process utilizes the Ion exchange column purification. In contrast, harvesting of viral vector genome from lymphocytes is performed using the kits designed for the Extraction of DNA from Blood and Tissues.

The AAV genomes are generally treated with DNAase- I at 10 units/ml in presence of 0.05% plutonic F68 (Invitrogen) at 37-degree celsius for 30 minutes to 60 minutes and diluted to within the linear range of the ddPCR assay (1 to 10e5 vg copies)

Preparation of Mastermix

• Prepare a reaction mixtures following the ddPCR supermix for Probes (Bio-Rad, Hercules, CA) kit protocol.
• The final concentration of TaqMan primers and probes used in the qPCR genome titer assays are 900 and 250 nM, respectively
• Add 5 μl of template DNA and adjust a 20 μl final volume of each reaction

Generation of Droplet and Analysis

• Load each reaction into the sample well of eight well disposable along with70 μl of droplet generation oil (Bio-Rad), and generate droplets in a droplet generator (Bio-Rad). 
• Transfer droplets to a 96-well PCR plate, heat-sealed with foil, and amplify in a conventional thermal cycler. 
• Thermocycler Typical Program Details

                95°C for 10 min, followed by 42 cycles of 95°C for 30 sec, 

                60°C for 1 min, and 72°C for 15 sec followed by 

                a final 98°C heat treatment for 10 min 

• Analyze the PCR plate using a QX100 droplet reader (Bio-Rad) and QuantaSoft software (Bio-Rad). 

Representation of ddPCR readouts

Data Analysis

The copies per microliter readout from the QX100 reader should be converted to genome copies per milliliter according to the formula 

X=[(aY)(1000/b)]D, where 

    X is GC/ml, 

    a is the volume of the ddPCR (20 μl), 

    Y is ddPCR readout copies per microliter, 

    b is the volume of the diluted vector in the ddPCR (5 μl), and 

    D is a total dilution applied to the test material. 

Universal Precaution

• Work in a biosafety cabinet or HEPA-filtered PCR station with UV light capability, preferably a dedicated unit for ddPCR analysis.
• Wear gloves at all times and follow the aseptic condition (use Lysols, and 70% ethanol to wipe out working areas, instruments, and pipettes. 

Suggested Pages
Instrument & Technology For Digital Droplet PCR

References 

Lock et al. 2014 Hum Gene Ther Methods. 

Wang et al. 2020 Method & Clinical Development

Sanmiguel et al. 2019 Adeno Associate Virus  Vectors