Two-Step Reverse Transcription Droplet Digital PCR Protocols for SARS-CoV-2 Detection and Quantification

These protocols can be used to develop in-house multiplex ddPCR assays for detection of SARS-CoV-2 using a two-color ddPCR system. This is useful for people who want to develop their own kits for the detection of SARS-CoV-2 and other pathogens. Compared to the gold standard RT-qPCR, RT-ddPCR can be used to quantify multiple SARS-CoV-2 genes without the need of a standard curve.

Since ddPCR is an emerging technique, visual demonstration is important to help current and future users understand and easily develop multiplex assays for SARS-CoV-2 detection and other pathogens. For cDNA synthesis, add two microliters of 5X reverse transcription master mix, five microliters of the RNA sample of interest, and three microliters of RNase-free double distilled water into a 100 or 200 microliter PCR tube to a final volume of 10 microliters as solution, and place the tube on ice. When off the reagents have been added, load the sample into a thermal cycler and set the instrument to run a reverse transcription at 37 degrees Celsius for 15 minutes, a heat inactivation of the reverse transcription for 5 seconds at 85 degrees Celsius, and a final infinite hold at four degrees Celsius.

Before performing droplet digital PCR, add 19.8 microliters of freshly prepared master mix to the appropriate number of wells of a nuclease free 96-well droplet digital PCR plate. Add 2.2 microliters of cDNA to each well of master mix to a final volume of 22 microliters of reaction mix per well and seal the plate with a disposable PCR plate sealer. Then, vortex the plate for 15 to 30 seconds in centrifuge for 10 to 15 seconds to collect the well contents at the bottom of the plate.

For automated droplet generation on the automated droplet generator touchscreen, click configure sample plate and select the columns in which the samples are located on sample plate. Click okay and open the automated droplet generator door. Load the sample and droplet generation plates and the other materials necessary for the experiment into the respective locations within the instrument and close the door.

When the touchscreen turns green, indicating that all of the materials have been loaded into their correct positions, select oil for probes and start droplet generation. Wait for the droplet generation to run to completion according to the time indicated on the screen. When the screen says Droplets ready, open the door to remove the plate containing the droplets and use a plate sealer for five seconds at 180 degrees Celsius to seal the plate with a pierceable foil seal.

Within 30 minutes of the droplet generation, place the sealed droplet plate into a 96 deep well thermal cycler. Set the sample volume to 40 microliters and the lid temperature to 105 degrees Celsius. Then, PCR amplify the droplets.

For droplet reading, after the amplification, transfer the thermal cycled 96-well plate into a droplet reader and open the accompanying software on a computer connected to the droplet reader. From the setup mode, select New and double click on any well to open the well editor dialogue box. Select the wells to be read and set experiment to absolute quantification, supermix to droplet digital PCR supermix for probes.

Target one type to channel 1 unknown and target two type to channel 2 unknown. Assign the sample names based on the plate layout and click apply and okay. After saving the template, click Run.

When prompted by the software, select the FAM/HEX color. At the end of data acquisition, remove the plate from the reader. Before analyzing the data, check the data from all of the wells to obtain the total number of droplets.

And set a cutoff to accept a minimum number of droplets to be counted as positive or negative results. For simplex and duplex assay analysis, double click on the QLP file to be analyzed and select Analyze. Use the 1D amplitude and 2D amplitude threshold tools to distinguish between the positive and negative droplets for each well in the correct channel, using the node template control and positive control samples as a guide.

Then, export the results as a CSV file for additional analysis. For a triplex probe mix assay, open the QLP file and select the wells to be analyzed in the plate editor tab. Set the experiment type to direct quantification and the assay to probe mix triplex, and enter the target name.

Then click Apply. Use the graph tools to assign specific colors to different target clusters as directed by the select to assign cluster window popup suggestions. When all of the target colors have been assigned, the quantification data will be displayed in the well data window.

Export the data as a CSV file for downstream analysis. For a quadruplex assay, set the experiment type to direct quantification and the assay to amplitude multiplex. Enter the target name and click Apply.

Use the graph tools to assign specific colors to different target clusters as directed by the select to assign cluster window popup suggestions. When all of the target colors have been assigned, the quantification data will be displayed in the well data window. Export the data as a CSV file for additional analysis.

As observed in this representative temperature gradient analysis, higher kneeling temperatures could not clearly distinguish positive droplets from negative droplets when a duplex assay was run. However, with a decrease in a kneeling temperature, an optimal separation between positive and negative droplets was achieved. Using a two color RT droplet digital PCR detection system, it is possible to detect one, two, three, and four SARS-CoV-2 targets within a single sample.

A no template control sample can be used to locate negative droplets to help set the negative threshold in simplex assays. In duplex assays, the analysis can be performed in individual channels or in the 2D amplitude. Although higher order multiplex assays data analysis is not straightforward, these assays are useful, because they allow up to eight droplet clusters to be assessed in triplex probe mix assays and up to 16 droplet clusters for quadruplex amplitude-based assays.

Depending on the assay to be developed, make sure the correct target primer and probe concentrations are added to the master mix. Using this procedure, one may alternate the targets to develop novel RT-ddPCR assays. This assays can be used for different research purposes or for the diagnosis of emerging infectious diseases.

Since its development, this technique has been used for the accurate determination of viral nucleic acid copies in different standards, the human body, and environmental samples.