TITLE: Extraction of Total Nucleic Acid from Wastewater Using the Promega Wizard Enviro Total Nucleic Acid Kit
AUTHORS: Tamara  Walsky, Padmini   Ramachandran, Amanda  Windsor, Maria  Hoffmann, Chris   Grim

[DESCRIPTION]
Wastewater based epidemiology has proven to be a useful tool in the COVID-19 pandemic, allowing prevalence and variant and sub-lineage surveillance on a watershed population level in a non-intrusive manner.  Essential to this method is the efficient extraction of viral genomic material from wastewater.  The process of detecting SARS Co-V-2 genetic signatures in wastewater samples involves collection of water, either as a grab sample or as a 24-hour composite sample, followed by sample concentration. Target genetic material may be present at a low concentration in water samples,  making sample concentration a prerequisite for sensitive detection. Concentration of microbial matter can be performed using a variety of methods, such as charged membrane filtration, centrifugal ultrafiltration and flocculation/precipitation using skim milk or polyethylene glycol (PEG)/NaCl. Most of the concentration methods were originally developed to concentrate live matter with the objective of culturing for detection of intact particles, though they have also been used for PCR-based detection. These methods have proven to be inconsistent, labor intensive and time consuming.  

More and more SC2-wastewater specific methods have recently been developed.  We have assessed the Promega Wizard Enviro Total Nucleic Acid kit for extraction of RNA for downstream SC2 detection and sequencing.  Briefly, the method directly captures and concentrates total nucleic acids (TNA) from a large volume of water using PureYield™ columns. The method uses a short protocol that minimizes the need for specialized laboratory equipment. In a first step total nucleic acid from a large volume sample (e.g., 40ml of wastewater) is captured on a PureYield™ Binding Column and then eluted in 1ml. In a second step, the material is further purified and concentrated using the PureYield™ Minicolumn. This method achieves consistent recovery rates and significant reduction in PCR inhibitors.

The total nucleic acid extracted using this kit can be analyzed for SARS-CoV-2 targets using a SARS-CoV-2 RT-qPCR
kit for wastewater. Please visit the Promega website for more information on these products:
https://www.promega.com/applications/infectious-diseases/covid19-wastewater-sars-cov-2-detection/.  Additionally, the extracted RNA can be utilized for SC2 variant sequencing: Enhanced QIAseq DIRECT SARS-CoV-2 Kit for Illumina MiSeq (protocols.io), or Modified NEBNext® VarSkip Short SARS-CoV-2 Library Prep Kit for Illumina Platforms - adapted for wastewater samples (protocols.io).

[BEFORE_START]
Prepare the following solutions prior to beginning nucleic acid extraction in Section 4:

Column Wash 1 (CWE): Add 57 mLof isopropanol to the Column Wash 1 (CWE) bottle and mark on the bottle “plus
isopropanol”. This reagent is stable at 15 °C to 30 °C when tightly capped.

Column Wash 2 (RWA): Add 350 mLof 95 % (v/v) ethanol the Column Wash 2 (RWA) bottle and mark the bottle “plus ethanol”. This reagent is stable at 15 °C to 30 °C when tightly capped.

Turn on water bath to 60 °C 


[STEPS]
SECTION: Capture and Concentration
1. Dispense 40 mL  of wastewater or pasteurized wastewater into a 
 

 


SECTION: Capture and Concentration
2. Add 500 µL  of Protease Solution.  Mix well by inversion and incubate for 30 min at Room temperature .

SECTION: Capture and Concentration
3. 3000 x g, 10 min, 25 Room temperature .
 


SECTION: Capture and Concentration
4. Carefully decant 20 mLof the supernatant into each of two clean
 
Discard the 50ml conical tube containing the pellet into an appropriate biohazard waste container.
 

 

SECTION: Capture and Concentration
5. To each tube containing 20 mLof the clarified supernatant, add 6 mLof Binding Buffer 1 (BBD) followed by
500 µLof Binding Buffer 2 (BBE).

SECTION: Capture and Concentration
6. Mix well by inverting the tube gently 10 times, or until thoroughly mixed.
SECTION: Capture and Concentration
7. Add 24 mLof isopropanol to each tube.

SECTION: Capture and Concentration
8. Mix well by inverting the tube gently 10 times, or until thoroughly mixed.
SECTION: Capture and Concentration
9. Setup the vacuum manifold as follows (see Figure 1): remove vacuum port cap, attach a Reservoir Extension Funnel to the PureYield™ Binding Column, then connect the column to the vacuum manifold by pressing the nozzle gently into the vacuum port.  

 

 

SECTION: Capture and Concentration
10. Pour the mixture from each tube from Step 8 into the Reservoir Extension Funnel on the PureYield™ Binding
Column (combine both tubes of the same sample if applicable), turn on the pump and apply vacuum to capture
TNA on the column.
 

SECTION: Capture and Concentration
11. Add 5 mLof Column Wash 1 (CWE) and apply a vacuum to pull the liquid through the PureYield™ Binding
Column.
 

SECTION: Capture and Concentration
12. Add 20 mLof Column Wash 2 (RWA) and apply a vacuum to pull the liquid through the PureYield™ Binding
Column. Continue to draw a vacuum for an additional 30 s after all visible liquid has passed through the
membrane.
 

SECTION: Capture and Concentration
13. Release the vacuum and remove the column from the vacuum manifold. Preheat 1.2 mLof Nuclease-Free Water,
per sample, to 60 °C for 5 min.

SECTION: Capture and Concentration
14. Assemble the elution device by placing a 1.5ml microcentrifuge tube into the base of the Eluator™ Vacuum
Elution Device (Cat.# A1071) and securing the tube cap in the open position, as shown (Figure 2).
Insert the PureYield™ Binding Column into the top of the Eluator Device, making sure the column is fully seated
on the collar as shown in Figure 3.
    

SECTION: Capture and Concentration
15. Place the Eluator™ Device assembly onto a vacuum manifold (Figure 3). Add 500 µLof preheated (60 °C)
Nuclease-Free Water to the PureYield™ Binding Column. Apply maximum vacuum for 1 minor until all
liquid has passed through the column. Repeat the process by adding another 500 µLof preheated Nuclease-Free
Water to the PureYield™ Binding Column to elute a total of 1 mLof TNA solution.

SECTION: TNA Extraction and Clean-Up
16. Add 400 µLof Binding Buffer 1(BBD) and 100 µLof Binding Buffer 2 (BBE) to 1 mLof liquid eluted in 
Step 15.
SECTION: TNA Extraction and Clean-Up
17. Mix well by inverting the tube gently 10 times, or until thoroughly mixed.  Divide the contents into two 
 
containing 750 µLeach.

SECTION: TNA Extraction and Clean-Up
18. 1 min Add 750 µLof isopropanol to each tube and mix well by inverting the tube gently 10 times, or until thoroughly mixed.

SECTION: TNA Extraction and Clean-Up
19. Place the PureYield™ Minicolumn into a PureYield™ Collection Tube. Pass the entire volume of the mixture
through the column, 750 µLat a time (a total of four times), using a microcentrifuge at 10.000 rpm, 1 min.

SECTION: TNA Extraction and Clean-Up
20. Add 300 µLof Column Wash 1 (CWE) and pull through the PureYield™ Minicolumn by centrifugation 10000 rpm, 1 min . Discard the flow-through.
SECTION: TNA Extraction and Clean-Up
21. Add 500 µLof Column Wash 2 (RWA) and pull through the PureYield™ Minicolumn by centrifugation 10000 rpm, 1 min . Repeat this wash one time. Discard the flowthrough.
SECTION: TNA Extraction and Clean-Up
22. Centrifuge 10000 rpm, 30 s to remove any residual wash solution.

SECTION: TNA Extraction and Clean-Up
23. Preheat 65 µLof Nuclease-Free Water per sample to 60 °C for 5 min.

SECTION: TNA Extraction and Clean-Up
24. Transfer the PureYield™ Minicolumn to a new 
 
and add 30 µLof preheated (60 °C) Nuclease-Free Water to the column. Let the water soak into the column filter for approximately 1 min.

SECTION: TNA Extraction and Clean-Up
25. Centrifuge 10000 rpm, 1 min to elute. Repeat elution with another 30 µLof preheated Nuclease-Free Water, for a total of 60 µL. 

SECTION: TNA Extraction and Clean-Up
26. Store sample at or below -20 °C until further analysis. TNA purified using this method can be directly used for
RT-qPCR, for example as input into the following protocol: 
RTqPCR of SARS-CoV-2 N1 Target on ABI 7500 Fast Using Promega GoTaq Enviro Wastewater SARS-CoV-2 System  V1 (protocols.io)