Quantification and Whole Genome Characterization of SARS-CoV-2 RNA in Wastewater and Air Samples

Wastewater-based epidemiology is currently used to complement COVID-19 control strategies worldwide by detecting viral RNA in wastewater samples and identifying potential outbreaks. This technique provides a population-wide snapshot of the prevalence of COVID-19 in our community, which includes asymptomatic cases. Air sampling is also an important addition to this protocol.

Clinical diagnostics is still the most accurate way to manage disease outbreaks and pandemics. However, this technique is still useful as a complement to it. This method has many steps and critical points.

A visual demonstration is essential, as it will allow laboratories that are not so familiar with molecular biology to replicate the procedure. To begin, collect one liter of a 24-hour composite wastewater sample. Spike 20 microliters of mengovirus into 70 milliliters of the sample.

After centrifuging the sample at 700G for 10 minutes, concentrate the resulting supernatant using a centrifugal ultrafiltration device with a cutoff of 10 kilodaltons by centrifugation. Elute the concentrate by centrifuging at 700G for 40 minutes, and inverting the position of the ultrafiltration device. Use the resulting concentrate for RNA extraction.

To collect the air samples, place a sterile cone in the air sampler before starting the collection. Once the sampling is over, extract RNA from the washed PBS of the cone using a commercial viral RNA extraction kit or an in-house method. Prepare the reaction master mix for each target, mengovirus, SARS-CoV-2, N1 and N2 genes in a clean hood in the reagent setup room.

Mix the primers-probe mix and enzyme by inverting five times or pulse vortexing. Keeping the microcentrifuge tubes in a cold rack, dispense 15 microliters of the mix into strip PCR tubes or a 96-well PCR plate placed on a cooling rack. Cover the plate and move it to the nucleic acid handling area.

Gently vortex an aliquot of the extracted RNA for five seconds. Pipette five microliters of the RNA to the reaction mix to make 25 microliters and start the RT-qPCR. In a PCR plate, pipette 16 microliters of each sample to its position on the plate, and add four microliters of five times reverse transcription or RT master mix.

Then start the PCR reaction. Next, prepare 10 micromolar dilutions of each primer set and the master mix for each set of primers. In a new PCR plate, pipette 20 microliters of the master mix, then five microliters of the RT sample to each mix.

After covering the plate, start the PCR reaction. Once the PCR gets over, spin down the plate at 1000 G for 15 to 30 seconds. For PCR purification, add 50 microliters of bead-based reagent to each well and mix by pipetting to proceed with the magnetic separation.

Remove the plate from the magnetic stand. Resuspend each pellet with 15 microliters of PCR grade water and incubate the plate at room temperature For two minutes. Place the plate back on the magnetic stand and allow the beads to pellet for two minutes.

Then carefully pipette 15 microliters of the supernatant from each sample to a new PCR plate. Next, for library preparation, add 1.75 microliters of reaction buffer from the DNA library preparation kit and 0.75 microliters of enzyme mix to each sample. Briefly vortex the covered plate and spin down at 1000G for 15 to 30 seconds.

Incubate for five minutes at 21 degrees Celsius and five minutes at 65 degrees Celsius. Pipette three microliters of PCR grade water for each sample to a new PCR plate. Add 0.75 microliters of the prepared samples and 1.25 microliters of barcodes for sequencing library preparation.

Then add five microliters of T4 DNA ligase master mix and mix well by pipetting. After spinning down the plate in the centrifuge, incubate the plate at 21 degrees Celsius for 20 minutes, and at 65 degrees Celsius for 10 minutes. After pooling all the samples, add 192 microliters of bead-based reagent to the pool for PCR purification.

Mix well by pipetting and incubate at room temperature for 10 minutes. Place the tube on a magnetic stand. Wait for the supernatant to clear and a bead pellet to form.

Remove the supernatant and the tube from the magnetic stand. Add 700 microliters of the short fragment buffer or SFB and mix by pipetting. Place the tube back on the magnetic stand and wait for the pellet to form and the liquid to clear.

Discard the supernatant using a pipet. Add 35 microliters of PCR grade water to the tube, mix by pipetting and incubate at room temperature for two minutes. Place the tube on the magnetic stand and allow the beads to pellet and the liquid to clear.

Pipette 35 microliters of the supernatant to a new tube. Next, prepare the adapter ligation reaction mix as described in the text manuscript, and incubate at room temperature for 10 minutes. Mix 20 microliters of the bead-based reagent to the ligation reaction mix for PCR purification, and incubate for 10 minutes at room temperature.

Place the tube on the magnetic stand and wait for the bead pellet to form and the supernatant to become colorless. Carefully discard the supernatant by pipetting. Mix 125 microliters of SFB into the tube and place it back on the magnetic stand to separate the beads.

When the liquid turns clear, discard the supernatant and repeat adding SFB as demonstrated previously. Leave the tube on the magnetic stand open for 30 seconds. Add 15 microliters of the elution buffer and mix well by pipetting.

After a brief spin down and incubation at room temperature for five minutes, place on the magnetic stand for two minutes. Pipette 15 microliters of the supernatant into a new low binding tube to measure the concentration with a fluorometric DNA quantification kit. Next, insert the flow cell into the real-time DNA and RNA sequencing device.

Insert the tip of a 1, 000 microliter pipette set to 200 microliters into the priming port. Turn the volume setting wheel to increase the volume until seeing the liquid in the tip, and then discard the tip. Add 800 microliters of the priming mix to the priming port without bubbles and wait five minutes.

Pipette 200 microliters of the library mix into the priming port. Close the cover port and priming port. Start the sequencing experiment in the software and select Base Calling On.This method detected and quantified SARS-CoV-2 RNA in wastewater and air samples.

Wastewater samples had a standard deviation from 1.91%to 13.98%among the triplicates, and ranged from 3.05 times 10 to the three to 2.83 times 10 to the eight gene copies per liter. Air samples ranged from 6.17 times 10 to the three to 5.48 times 10 to the nine, with the standard deviation in the duplicates between 0.54%and 10.95%As described in this protocol, the samples were sequenced, and an example of three sequenced sample results is summarized here. In all three samples, the lineage BA.5.

x was assigned as the most prevalent by the Freyja tool. The concentration of wastewater samples is a crucial step as viral RNA is present at low concentrations in wastewater. And so this step is fundamental for accurate detection and quantification.

This procedure can be adapted by using different primers in the RT-qPCR protocol to be used to detect and quantify other infectious agents. The technique accurately quantified SARS-CoV-2 in wastewater and air samples. Different pathogens can be detected and quantified from different locations.