May 31, 2022
Extraction of Total Nucleic Acid from Wastewater Using the Promega Wizard Enviro Total Nucleic Acid Kit
- 1FDA CFSAN
Protocol Citation: Tamara Walsky, Padmini Ramachandran, Amanda Windsor, Maria Hoffmann, Christopher Grim 2022. Extraction of Total Nucleic Acid from Wastewater Using the Promega Wizard Enviro Total Nucleic Acid Kit. protocols.io https://dx.doi.org/10.17504/protocols.io.4r3l2oebxv1y/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: May 13, 2022
Last Modified: May 31, 2022
Protocol Integer ID: 62567
Keywords: wastewater, total nucleic acid, nucleic acid extraction
Abstract
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).
Materials
Wizard® Enviro Total Nucleic Acid Kit, Promega cat# A2991
Contains sufficient materials and reagents for 25 samples. Includes:
• 1 × 13ml Protease Solution
• 1 × 320ml Binding Buffer 1 (BBD)
• 1 × 30ml Binding Buffer 2 (BBE)
• 1 × 85.3ml Column Wash 1(CWE)
• 1 × 206ml Column Wash 2 (RWA)
• 1 × 150ml Nuclease-Free Water
• 5 × 5 each PureYield™ Binding Columns
• 1 × 25 each PureYield™ Minicolumns
• 1 × 25 each PureYield™ Collection Tubes
• 1 × 50 each Elution Tubes
• 1 × 25 each Reservoir Extension Funnel
Storage Conditions: Store all components at +15°C to +30°C.
Materials to Be Supplied by the User
• isopropanol
• ethanol, 95%
• tabletop centrifuge (capable of 3,000 × g)
• swinging bucket rotor (that accommodates 50ml tubes)
• 50ml disposable plastic screw-cap tubes (e.g., Corning® or Falcon® brand)
• 1.5ml microcentrifuge tubes
• heat block (capable of reaching 60°C)
• high-speed microcentrifuge (for tubes, capable of at least 10,000rpm)
• vacuum manifold (e.g., Vac-Man® Laboratory Vacuum Manifold, Cat.# A7231)
• Eluator™ Vacuum Elution Device (Cat.# A1071)
• vacuum pump, single- or double-stage, producing pressure of approximately 650mm Hg (25.6 inches Hg,
12.57psi, 86.7kPa).
Before start
Prepare the following solutions prior to beginning nucleic acid extraction in Section 4:
Column Wash 1 (CWE): Add 57 mL of 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 mL of 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
Capture and Concentration
Capture and Concentration
Dispense 40 mL of wastewater or pasteurized wastewater into a
Equipment
50 mL conical screw-cap tube
NAME
Plastic consumables
TYPE
Grenier Bio-One
BRAND
5622-7261
SKU
LINK
Any brand or size of plastic tube with a tight-fitting screw cap will work fine.
SPECIFICATIONS
Note
Pasteurizing wastewater is optional. To pasteurize wastewater, incubate at60 °C for 01:00:00 Please follow your institution’s biosafety guidelines.
Add 500 µL of Protease Solution. Mix well by inversion and incubate for 00:30:00 at Room temperature .
30m
3000 x g, Room temperature, 00:10:00 .
Note
This step is designed to remove solids, to avoid clogging the PureYield™ Binding Column.
10m
Carefully decant 20 mL of the supernatant into each of two clean
Equipment
50 mL conical screw-cap tube
NAME
Plastic consumables
TYPE
Grenier Bio-One
BRAND
5622-7261
SKU
LINK
Any brand or size of plastic tube with a tight-fitting screw cap will work fine.
SPECIFICATIONS
Discard the 50ml conical tube containing the pellet into an appropriate biohazard waste container.
Note
Sometimes, the solids will produce a loose pellet. In this case, let the solids settle again before decanting or use a serological pipet to avoid transferring solids to the binding column.
Note
If you wish to process the pelleted solids to collect additional total nucleic acid, see Section 5.A. of the Wizard® Enviro Total Nucleic Acid Kit Technical Manual.
To each tube containing 20 mL of the clarified supernatant, add 6 mL of Binding Buffer 1 (BBD) followed by
500 µL of Binding Buffer 2 (BBE).
Mix well by inverting the tube gently 10 times, or until thoroughly mixed.
Add 24 mL of isopropanol to each tube.
Mix well by inverting the tube gently 10 times, or until thoroughly mixed.
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.
Figure 1. The Reservoir Extension Funnel and PureYield™ Binding Column attached to a Vac-Man®
Vacuum Manifold port. See Quick Start Guide for Assembly of a Vacuum Apparatus with the Welch Vacuum Pump
Technical Bulletin #TB355 for setup details.
Note
Using the Reservoir Extension Funnel allows up to 100 mL of sample mixture to be added to the PureYield™ Binding Column at one time.
Note
Due to the size of the assembled binding column and funnel, we suggest processing 6 to 8 samples at a time per vacuum manifold.
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.
Note
Periodically, empty the liquid waste collected in the blue Vac-Man® Laboratory Vacuum Manifold (Cat.# A7231). Dispose of the alcohol-containing waste following your institutional policies.
Add 5 mL of Column Wash 1 (CWE) and apply a vacuum to pull the liquid through the PureYield™ Binding
Column.
Note
Ensure that 57 mL of isopropanol has been added to the Column Wash 1 (CWE) bottle prior to use as described in the "Before Start" section.
Add 20 mL of 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 00:00:30 after all visible liquid has passed through the
membrane.
Note
Ensure that 350 mL of 95 % (v/v) ethanol has been added to Column Wash 2 (RWA) bottle
prior to use as described in the "Before Start" section.
30s
Release the vacuum and remove the column from the vacuum manifold. Preheat 1.2 mL of Nuclease-Free Water,
per sample, to 60 °C for 00:05:00 .
5m
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.
Figure 2. A 1.5ml microcentrifuge tube is placed in the base of the Eluator™ Vacuum
Elution Device with the tube cap locked as shown.
Figure 3. The final Eluator™ Vacuum Elution Device assembly, including the binding column, ready for use on a vacuum manifold.
Place the Eluator™ Device assembly onto a vacuum manifold (Figure 3). Add 500 µL of preheated (60 °C )
Nuclease-Free Water to the PureYield™ Binding Column. Apply maximum vacuum for 00:01:00 or until all
liquid has passed through the column. Repeat the process by adding another 500 µL of preheated Nuclease-Free
Water to the PureYield™ Binding Column to elute a total of 1 mL of TNA solution.
1m
TNA Extraction and Clean-Up
TNA Extraction and Clean-Up
11m 30s
11m 30s
Add 400 µL of Binding Buffer 1(BBD) and 100 µL of Binding Buffer 2 (BBE) to 1 mL of liquid eluted in
Step 15.
Mix well by inverting the tube gently 10 times, or until thoroughly mixed. Divide the contents into two
Equipment
DNA LoBind Tube 1.5 mL
NAME
Microcentrifuge tube
TYPE
Eppendorf
BRAND
022431021
SKU
LINK
containing 750 µL each.
00:01:00 Add 750 µL of isopropanol to each tube and mix well by inverting the tube gently 10 times, or until thoroughly mixed.
1m
Place the PureYield™ Minicolumn into a PureYield™ Collection Tube. Pass the entire volume of the mixture
through the column, 750 µL at a time (a total of four times), using a microcentrifuge at 10.000 rpm, Room temperature, 00:01:00 .
1m
Add 300 µL of Column Wash 1 (CWE) and pull through the PureYield™ Minicolumn by centrifugation 10000 rpm, Room temperature, 00:01:00 . Discard the flow-through.
1m
Add 500 µL of Column Wash 2 (RWA) and pull through the PureYield™ Minicolumn by centrifugation 10000 rpm, Room temperature, 00:01:00 . Repeat this wash one time. Discard the flowthrough.
1m
Centrifuge 10000 rpm, Room temperature, 00:00:30 to remove any residual wash solution.
30s
Preheat 65 µL of Nuclease-Free Water per sample to 60 °C for 00:05:00 .
5m
Transfer the PureYield™ Minicolumn to a new
Equipment
DNA LoBind tubes, 1.5 mL
NAME
Tubes
TYPE
Eppendorf
BRAND
022431021
SKU
LINK
1.5 mL
SPECIFICATIONS
and add 30 µL of preheated (60 °C ) Nuclease-Free Water to the column. Let the water soak into the column filter for approximately 00:01:00 .
1m
Centrifuge 10000 rpm, Room temperature, 00:01:00 to elute. Repeat elution with another 30 µL of preheated Nuclease-Free Water, for a total of 60 µL .
1m
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: