I have seen some papers where they don't lyse:
Absolute Determination of Single-Stranded
and Self-Complementary Adeno-Associated
Viral Vector Genome Titers by Droplet Digital PCR. Jim Wilson. 2014.
But some kits (Takara and ABMgood) have a "virus lysis solution". Does anyone know if the lysis step is necessary?
The initial 94 degrees C step of the PCR should be enough to denature the viral capsid, so no lysis is necessary and you can add the viral vector directly to the PCR. See a protocol here: https://www.addgene.org/protocols/aav-titration-qpcr-using-sybr-green-technology/
Before you can claim that it does not make a difference if you lyse before or during the PCR cycles, one should carefully validate if this is true. Weird things can happen in a reaction tube that don't fit to the theory on paper.
So, make sure that the lysis by heating the capsids really can release 100% of DNA molecules and if all of those are detected, which can be different for qPCR and ddPCR. This sounds easier than it is done (e.g. DNA release by heating could have different efficiency for different serotypes).
One more thing that is rarely mentioned with regard to ddPCR:
Some or most people do the lysis of capsids before they generate droplets, not considering the fact that the AAV ssDNA genomes are 50% plus and 50% minus strands. Therefore, your DNA can anneal after lysis and form dsDNA which end up in one droplet leading to 1 copy counted instead of initially 2 copies. This process can be time- and concentration-dependent, so you can get anything between 50-100% of the initial copy number of ssDNA containing capsids.
If you don't validate your process, the advantage of ddPCR (no standard curve needed) can turn into a disadvantage because there is no control for correctness of your result. You just have an "absolute" copy number with less variation between experiments compared to qPCR (which is nice) suggesting an accuracy or correctness which it does not have unless you validate the method properly.
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