High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses
Aaron Topol, Marlene Wolfe, Brad White, Krista Wigginton, Alexandria B Boehm
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Abstract
This process instruction describes the steps for pre-analytical processing of primary settled solids from wastewater treatment plants for downstream nucleic acid purification and quantification. Previous research has demonstrated that enveloped viral particles, such as SARS-CoV-2, associate with the solids in wastewater. Therefore, concentrating the solids in the sample and removing the water concentrates the viral particles as well and increases the sensitivity of the assay.
Bovine coronavirus vaccine (BCoV) is spiked into samples before nucleic acid extraction and serves as a process control as well as an indicator of PCR inhibition.
The dry weight of the sample is determined to account for sample variability between different treatment plants and between samples collected on different days and allows for the final quantification to be normalized to the precise quantity of solids in the sample. A dry weight conversion factor to adjust for the final amount of solids in the quantified sample is determined by measuring the difference in mass between the “wet” dewatered solid and after the sample is dried at 110°C overnight.
For long term storage, up to 50mL of the original primary settled solid sample is stored at 4°C and small aliquot of the dewatered solids is stored at -80°C.
This process instruction applies to sample dewatering, BCoV control spike in, homogenization, dry weight measurement, aliquoting and storage.
Steps
Preparation
Process fresh samples stored at 4°C if possible. If analyzing a frozen sample, thaw at 4Room temperature . * Set centrifuge temperature to 4°C.
- Set oven temperature to
110°C. - Remove an aliquot of BCoV from the freezer and thaw
110On ice.
Dewater Sludge Samples by Centrifugation
Label 50 mL conicals with the sample name.
Invert the sludge sample bottle a few times to mix.
In a biosafety cabinet, pour 50mL of well mixed primary sludge sample to corresponding labeled 50 mL conical. If the total volume of a sample is less than 50 mL, bring the volume up to 50 mL with nuclease free water.
Bring the rotor to a biosafety cabinet and arrange the conicals in the rotor in a manner to ensure that it is balanced.
Centrifuge samples at 24000x g,0h 0m 0s for at least 0h 30m 0s at 4°C. Set the Centrifuge time to “Hold”.
In a biosafety cabinet, aspirate supernatant into a liquid biohazard waste container.
BCoV Spike-in and Sample Homogenization
Apply a label to a new 50 mL conical for each sample and record the empty conical mass in grams.
With a spatula (either plastic or metal), scoop a small aliquot of dewatered pellet (~0.75 g) into the new, pre-weighed 50 mL conical. Record the mass in grams of the aliquot in the conical. Set the rest of the pellet aside for dry weight measurement.
In a new 150 mL bottle, prepare a stock solution of DNA/RNA shield with BCoV spike-in by adding 1.5µL rehydrated BCoV vaccine per 1mL DNA/RNA shield. Mix well by inverting or gently shaking the bottle.
Add DNA/RNA shield with BCoV spike-in to each conical containing solids so that the final concentration of dewatered solids resuspended in the BCoV-spiked DNA/RNA shield is ~
75mg/mL
Save the remaining DNA/RNA shield with BCoV spike-in to use as template in Extraction Positive Control during RNA Extraction.
Add 5-10 grinding balls to each conical tube.
Place the conicals in the adapters on the stage of the Geno/Grinder and tightly secure the clamp.
Equipment
| Value | Label |
|---|---|
| Geno/Grinder | NAME |
| Automated Tissue Homogenizer and Cell Lyser | TYPE |
| SPEX Sample Prep | BRAND |
| 1006-113806 | SKU |
| https://www.thomassci.com | LINK |
Shake the conicals in the Geno/Grinder for 0h 2m 0s at 1,000 rpm to homogenize the sample.
Briefly centrifuge the conicals for 0h 5m 0s at 5250x g,0h 0m 0s to pull down the bubbles generated during homogenization. Sample is ready for RNA extraction using the Chemagic 360 (see accompanying protocol, extraction may also be done using Kingfisher or several manual Qiagen kits. See protocol for details) .
Dry weight measurement
Place aluminum weigh boats in oven to pre-heat to 110°C.
Weigh each pan and record the mass in grams of each empty pan.
Using a spatula, scoop a generous amount of dewatered solid into the pan.
Record the mass in grams of each pan with the wet solids.
Place the dishes in the oven inside the biosafety cabinet and leave overnight to dry,
The next day or at least 6 hours later (6h 0m 0s - 24h 0m 0s dry time), remove the pans from the oven and record the mass in grams.
Dispose of pans in the red biohazard waste bin.
Aliquot and Sample Storage
Apply a label for each sample to a 50 mL conical.
Pour up to 50mL of remaining primary settled solids sample and place at 4°C for storage until it is confirmed that results of the analyses pass quality control. Dewatered solids can be stored at -80 deg C. We recommend that you confirm the effects of storage on target quantification. We have noted that up to 7 d storage at 4 deg C has minimal affect on the RNA targets. However, freeze thaws may affect target quantification.
Dispose of remaining sludge in 4 L Biohazard waste container prefilled with 400mL of bleach. Then dispose of the sample bottles as biohazard waste.
