To begin, take the thawed droplet digital, or ddPCR master mix and add probes, forward primer, and reverse primer to it at room temperature. Prepare the master mix for each amplification target following the suggested composition shown on the screen, and adjust the concentrations of primers and probes as needed. Vortex the mixture thoroughly and briefly centrifuge it in a mini centrifuge.
Add the restriction enzyme and invert to mix. Next, add 16.5 microliters of the prepared master mix to each well of a PCR plate and seal the plate with a transparent adhesive film. Using the sample dilution buffer as the diluent, prepare quality control, or QC dilutions as outlined in this table which represents the recommended concentrations for a validation accuracy and precision run.
Dilute the tear samples with a sample dilution buffer at a 1-to-10 ratio in 0.2-milliliter PCR tubes and seal the tubes. Heat the samples in a thermocycler at 95 degrees Celsius for 10 minutes and then at 4 degrees Celsius for at least 5 minutes. Remove the adhesive film from the ddPCR plate containing the master mix and add 5.5 microliters of QC dilution to appropriate wells of the 96-well plate as shown in the plate map.
Then add 5.5 microliters of sample to the wells. Add 5.5 microliters of sample dilution buffer to the no template control, or NTC wells. Dilute 2x ddPCR buffer control at a 1-to-2 ratio using nuclease-free water and add 22 microliters of the buffer to any empty wells of a column.
Add a pierceable foil seal to the plate and place the plate in the plate sealer. Seal the plate for 5 seconds at 180 degrees Celsius. Vortex the plate at the maximum speed for at least 30 seconds and centrifuge briefly in a plate spinner.
On the touchscreen of the Automated Droplet Generator, select the columns on the plate map containing the samples. The deck of the instrument will light up to indicate which consumables are required. Load the droplet generator from back to front.
The yellow light will change from yellow to green to indicate the sufficiency of the consumables. Place a cold block and the droplet plate holder. Ensure the block is fully blue colored and no pink is visible.
Place a new 96 weld ddPCR plate in the cold block. Place the prepared PCR plate in the sample plate holder. Close the machine lid, and press start for droplet generation.
Within 30 minutes, remove the plate containing the droplets from the cold block. Add a pierceable foil seal to the plate and place the plate in the plate sealer. Seal it for five seconds at 180 degrees Celsius.
Then place the plate in a compatible thermal cycler and enter the cycling conditions shown on the screen. Load the plate into the droplet reader ensuring sufficient reader oil remains and the waste container has sufficient space. Finally, press the read button on the software to start reading the droplets.
An intra assay mean for each concentration in each assay was calculated and this was used to assess the precision and accuracy of the assay. A mean inter-assay precision of 7.7%and a mean absolute inter-assay accuracy of 4.2%were found across all QC levels. Inter-assay accuracy and precision were also calculated using the inter-assay mean of each QC level within each batch.
An inter-assay precision ranging from 4-to-8.5%and an inter-assay absolute accuracy ranging from one to 3.2%were found. Similarly, for the tear sample a mean inter-assay precision of 3.7%at the high spike level and 12.2%at the low spike level and a mean inter-assay absolute accuracy of 11.3%at the high level and 8.1%at the low level were observed. In the case of inter assay calculation, a precision of 5.5%at the high level and 7.1%at the low level and an absolute accuracy of 11.3%at the high level and 8.1%at the low level were found.
