Digital Droplet PCR Assay to Measure AAV vector DNA

Droplet digital PCR (ddPCR):

A third generation of quantitative PCR, water and oil emulsions is used to segment the samples and create droplets to identify and quantify the genetic material in the sample. It 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; all properties that lend themselves to the accurate quantification of both single-stranded and self-complementary AAV genomes.

The advantage of ddPCR for the nucleic acid quantification includes: 

• Unparalleled precision: the massive sample partitioning afforded by ddPCR enables small fold decrease in target DNA sequences between samples to be reliably measured.  
• Increased signal-to-noise: enrich for rare targets by reducing competition that comes from high-copy templates. 
• Removal of PCR efficiency bias: error rates are reduced by removing the amplification efficiency reliance of PCR, enabling accurate quantification of targets.
• Simplified quantification: a standard curve is not required for absolute quantification

The procedure/steps involved for quantification of viral DNA using Bio-rad's ddPCR are:

i) Droplet Generation 

ii) PCR Amplification

iii) Droplet Reading 

iv) ddPCR Data Analysis 

i) Droplet Generation

Before droplet generation, ddPCR reaction are prepared in a similar manner as read-time PCR reactions that use Taq man hydrolysis probes labeled with FAM and HEX (or VIC) reporter flurophores, or an intercalating dye such as EvaGreen.

• For the droplet generation, assemble the recommended ddPCR supermix (Bio-Rad, Hercules, CA) for preparing the reaction mixtures. 
• Use the pre-designed TaqMan primers and probes in final optimized concentrations of (~100 nM to 100 nM), and template (5 μl) in a final volume of 20 μl. 
• Load each reaction into the sample well of an eight-well disposable cartridge (Bio-Rad) along with 70 μl of droplet generation oil (Bio-Rad), and generate droplets in a droplet generator (Bio-Rad). 
• The droplet generator produces about 20,000 droplets per sample in about 2.5 min for eight samples. 

ii) PCR Amplification

• Gently pipette and transfer droplets to a 96-well PCR plate and seal the plate with PCR plate sealer and pierceable foil heat seal
• Amplify the DNA to the end point with a conventional thermal cycler with recommended cycling protocol (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).

iii) Droplet Reading

• Open the QuantaSoft software in the setup mode and design a new plate with a layout according to the experimental design.
• Designate the sample name, experiment type, corresponding fluorescence channels such as FAM and HEX.
• Scan the PCR plate on QX100 droplet reader (Bio-Rad).

iv) ddPCR Data Analysis 

• Analyze the data with QuantaSoft software.
• If the plate is set up for absolute quantitation analysis, automatic thresholding determines concentration and populates the data tables in the analysis mode of the software. 
• The concentration reported is coplies per microliter of the final 1X ddPCR reaction. 
• 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 volume of the ddPCR (20 μl),

        Y is ddPCR readout copies per microliter, 

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

        D is total dilution applied to the test material.



References:

Primer and Probe designing for ddPCR/qPCR
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3991984/

https://www.bio-rad.com/webroot/web/pdf/lsr/literature/Bulletin_6407.pdf