TITLE: High Throughput pre-analytical processing of wastewater settled solids for SARS-CoV-2 RNA analyses
AUTHORS: Aaron Topol, Marlene Wolfe, Brad White, Krista Wigginton, Alexandria B B Boehm

[DESCRIPTION]
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]
SECTION: Preparation
1. Process fresh samples stored at 4 °C if possible. If analyzing a frozen sample, thaw at 4 Room temperature . 
Set centrifuge temperature to 4 °C.
Set oven temperature to 110 °C.
Remove an aliquot of BCoV from the freezer and thaw on ice.
SECTION: Dewater Sludge Samples by Centrifugation
2. Label 50 mL conicals with the sample name.
SECTION: Dewater Sludge Samples by Centrifugation
3. Invert the sludge sample bottle a few times to mix.
SECTION: Dewater Sludge Samples by Centrifugation
4. In a biosafety cabinet, pour 50 mL 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.

SECTION: Dewater Sludge Samples by Centrifugation
5. Bring the rotor to a biosafety cabinet and arrange the conicals in the rotor in a manner to ensure that it is balanced.
SECTION: Dewater Sludge Samples by Centrifugation
6. Centrifuge samples at 24000 x g for at least 30 min at 4 °C. Set the Centrifuge time to “Hold”.

SECTION: Dewater Sludge Samples by Centrifugation
7. In a biosafety cabinet, aspirate supernatant into a liquid biohazard waste container.
SECTION: BCoV Spike-in and Sample Homogenization
8. Apply a label to a new 50 mL conical for each sample and record the empty conical mass in grams. 
SECTION: BCoV Spike-in and Sample Homogenization
9. 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.
 



SECTION: BCoV Spike-in and Sample Homogenization
10. In a new 150 mL bottle, prepare a stock solution of DNA/RNA shield with BCoV spike-in by adding 1.5 µL rehydrated BCoV vaccine per 1 mL DNA/RNA shield. Mix well by inverting or gently shaking the bottle. 
 
  
  

SECTION: BCoV Spike-in and Sample Homogenization
11. 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 ~ 75 mg/mL 


 

SECTION: BCoV Spike-in and Sample Homogenization
11.1. Save the remaining DNA/RNA shield with BCoV spike-in to use as template in Extraction Positive Control during RNA Extraction.
SECTION: BCoV Spike-in and Sample Homogenization
12. Add 5-10 grinding balls to each conical tube.
SECTION: BCoV Spike-in and Sample Homogenization
13. Place the conicals in the adapters on the stage of the Geno/Grinder and tightly secure the clamp.

 

SECTION: BCoV Spike-in and Sample Homogenization
14. Shake the conicals in the Geno/Grinder for 2 min at 1,000 rpm to homogenize the sample.

SECTION: BCoV Spike-in and Sample Homogenization
15. Briefly centrifuge the conicals for 5 min at 5250 x g 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). 
 



SECTION: Dry weight measurement
16. Place aluminum weigh boats in oven to pre-heat to 110 °C.

SECTION: Dry weight measurement
17. Weigh each pan and record the mass in grams of each empty pan. 
SECTION: Dry weight measurement
18. Using a spatula, scoop a generous amount of dewatered solid into the pan.
SECTION: Dry weight measurement
19. Record the mass in grams of each pan with the wet solids. 
SECTION: Dry weight measurement
20. Place the dishes in the oven inside the biosafety cabinet and leave overnight to dry,
SECTION: Dry weight measurement
21. The next day or at least 6 hours later (360 min - 1440 min  dry time), remove the pans from the oven and record the mass in grams.

 

SECTION: Dry weight measurement
22. Dispose of pans in the red biohazard waste bin.
SECTION: Aliquot and Sample Storage
23. Apply a label for each sample to a 50 mL conical.
SECTION: Aliquot and Sample Storage
24. Pour up to 50 mL 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. 




SECTION: Aliquot and Sample Storage
25. Dispose of remaining sludge in 4 L Biohazard waste container prefilled with 400 mL of bleach. Then dispose of the sample bottles as biohazard waste.