Digital droplet PCR for AAV quantification (2023)

Ever since Addgene started producing AAV vector preparations, we have been using quantitative PCR to determine the physical titer of our preparations. We repeat this assay frequently to confirm our results are accurate. But what if we could repeat this essay 10,000 times to ensure our titles are as accurate as possible? This is where Digital Droplet PCR (ddPCR) comes into play.

AAV titration with quantitative PCR

Before diving into the details of ddPCR, we should first note that quantitative PCR (qPCR) has been a powerful tool to quantify AAV for some time. IfAAV-Titration durch qPCRviral DNA is amplified and monitored in real time. When the PCR is complete, the results are analyzed by comparing the viral DNA fluorescence threshold to the fluorescence threshold of a standard - a series of reactions with known amounts of DNA. A reference to AAV, a known-title virus, may also be usedConfirm that the standard curve provides an accurate reading of your samples.

Of course, if qPCR were perfect, there would be no need for ddPCR. A problem with qPCR is that the results can vary by a factor of 2. This means that if you set up two identical assays on the same sample, you may end up with a 4 x 10 titer12Genome copies/mL or 8 x 1012genome copies/mL and both results would be valid. For this reason, the AAV reference is crucial. This will help you determine if your titles are within the expected range.

We have also found that it can be difficult to properly construct the standard curve for qPCR. Here at Addgene we use a linearized plasmid pattern. But even if the pattern is run correctly, it is unstable and may only work for a small number of qPCR runs before a new pattern is needed. For these reasons we are in the process of changing our titration method from qPCR to ddPCR.

Digital Droplet PCR (ddPCR) requires no standard or reference, saving you reagents (and time)!

Digital droplet PCR for AAV quantification (1)

(Video) Digital PCR Principle & Advantages

AAV titration with digital droplet PCR

In digital droplet PCR, a PCR reaction mix is ​​divided into approximately 20,000 droplets using water-in-oil emulsion technology. Each drop contains the components of the target DNA amplification. This partitioning reduces the number of PCR inhibitors per reaction and allows for improved product detection. As an added benefit, replicas are built into the technology. One well (with thousands of droplets) may be enough to capture the information needed for your PCR experiment.

The ddPCR-AAV titration process starts with virus dilution. It is important to note that the dynamic range of ddPCR is between 1 and 100,000 genome copies (GC) per reaction. Since AAV titles tend to be in the 10th12for 1013GC/mL, you must serially dilute your virus before adding your sample to a master mix. At Addgene we usually load 3 dilutions onto our ddPCR plate. Because the titer is unknown, each dilution must be within the assay range for a broad titer range. We typically dilute our samples from 1:6 million to 1:25 million.

After making the dilutions, they are transferred to a new plate containing the master mix containing primers and a ddPCR supermix. Note that the Supermix you use and the oil drops must be from the same manufacturer. Otherwise you will end up with poor drop quality.

Using a droplet generator, the sample and droplet-producing oil are moved through small channels to create a water-in-oil emulsion containing approximately 20,000 droplets. Each drop contains the necessary material for a mini-amplification reaction to take place.

Digital droplet PCR for AAV quantification (2)When the PCR is complete, a droplet reader extracts the droplets from the plate and measures the fluorescence amplitude of each droplet. The fluorescent droplets contain the amplified target sequence. After reading all drops, the software generates an image of the measured fluorescence amplitude for each drop in each well (Figure 2). A clean ddPCR should have a clear separation between positive (blue) and negative (grey) droplets. Blank control wells should have few positive drops. In the figure to the right, there is approximately 1 positive copy per microliter in the non-template control wells. You will also notice a reduction in the number of positive droplets as the dilutions increase.

The ddPCR software uses the ratio of positive to negative droplets to calculate sample concentration. This concentration can then be used to calculate the virus titer:

GC/ml = {[(R*C)(1000/V)]*D}

R = reaction volume

(Video) Introduction to Droplet Digital™ PCR: Workflow and Applications

C = copies/µl

V = volume of virus in the reaction mixture

D = virus dilution factor

Most droplet readers have a few channels for fluorescence detection, making it possible to measure the concentration of multiple targets simultaneously. You only need to use one for AAV titration. However, two channels can be usedexamine the integrity of the viral genome(Furuta-Hanawa et al., 2019).

Tips and tricks for performing ddPCR

Clean everything

You must have a clean workspace to titrate your AAV with ddPCR. Digital droplet PCR is a sensitive assay, so cross-contamination in a non-template control (NTC) well is common. Here are some steps you can take to get a clean NTC:

  • Have a dedicated bank with a dedicated set of pipettes for the ddPCR setup.
  • Prepare the master mix in an area separate from where you will prepare your sample dilutions.
  • Aliquot all your reagents into disposable tubes and get a new one for every setup.
  • Clean the bench and all supplies with 10% bleach.

pipette slowly

If you are using a manual drop generator, you must be careful when transferring your drops from the drop generator to the PCR plate. Maintaining a high droplet count is important for calculating the concentration of your sample.

Even if you are using an automated droplet generator, avoid pipetting too quickly when diluting virus. This reduces aerosols that could potentially lead to contamination.

Optimize your PCR

For a good starting point for using PCR parameters, seeLock's seminal article on AAV titration(Locket al., 2014).

(Video) Digital Droplet PCR (ddPCR) Technology

If you are having trouble getting a clear separation between positive and negative droplets with these parameters, you can try the following:

  • Increase the number of cycles. After additional rounds of amplification, your fluorescence amplitude will increase, resulting in greater separation of your positive and negative droplets. More than 50 cycles are not recommended.
  • Lower your ramp rate. A rate of 2°C/s is recommended to ensure an even temperature change between all drops, but you can go as low as 1°C/s.
  • Increasing the elongation time to 2 minutes and the denaturation time to 1 minute has been demonstratedIncrease droplet separation(Witte et al., 2016). This is especially important when the amplicon is larger than 150 base pairs.

Although we use ddPCR for AAV titration, there are many applications for the technology. Digital droplet PCR is useful for detecting low copy numbers. Because of this, it can be used to detect rare sequences and for single-cell analysis. It is also used to detect microbes. Some applications includeMeasurement of viable probioticseDetection of circulating pathogens.

With its many uses and ease of use, digital droplet PCR is becoming a popular choice for PCR experiments.

Digital droplet PCR for AAV quantification (3)


Furuta-Hanawa, Birei, Teruhide Yamaguchi, and Eriko Uchida. "2D Digital Droplet PCR as a Tool for Titer and Integrity Assessment of Recombinant Adeno-Associated Viral Vectors."human gene therapyund (2019). PubMedPMID: 31140327. PubMed CentralPMC-ID: PMC6707039.

Gobert, Guillaume and others. "Digital droplet PCR improves the absolute quantification of viable lactic acid bacteria in stool samples."Journal of Microbiological Methods148 (2018): 64-73. PubMedPMID: 29548643.

Lock, Martin et al. "Absolute determination of single-stranded and self-complementary adeno-associated viral vector genome titers by digital droplet PCR." Human Gene Therapy Methods 25.2 (2013): 115-125. PubMedPMID: 24328707. PubMed CentralPMC-ID: PMC3991984.

(Video) Foundational: Counting in ddPCR

Song, Neng and others. "Detection of Mycobacterium tuberculosis-specific circulating DNA by digital droplet PCR for vaccine evaluation in challenged monkeys and TB diagnosis." Microbes and Emerging Infections 7.1 (2018): 1-9. PubMedPMID: 29691363. PubMed CentralPMC-ID: PMC5915492.

Witte, Anna Kristina and others. "A systematic study of parameters affecting droplet precipitation in the Listeria monocytogenes prfA assay - Reducing ambiguous results in ddPCR."Plus one11.12 (2016): e0168179. PubMedPMID: 27992475. PubMed CentralPMC-ID: PMC5167268.

Additional resources on the Addgene blog

  • Read more aboutPolymerase chain reaction
  • Browse our blog posts on the topicviral vector logs and tips
  • Read all of oursviral vector blog posts

Resources on

  • learn oursProtocol for AAV titration by qPCR
  • Browse our collection ofProtocols related to viral vectors
  • learn more aboutAddgene viral service

Subjects:viral vectors,Viral vector logs and tips,AAV

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