Apr 05, 2024
Microbe/Phage Wastewater DNA/RNA Concentration and Extraction (Nanotrap® and NucleoMag® RNA Water)
- Brett Rasile1,
- Kendra Maas1,
- Archana Anand2,
- Michael Hajkowski2
- 1University of Connecticut;
- 2San Francisco State University
Protocol Citation: Brett Rasile, Kendra Maas, Archana Anand, Michael Hajkowski 2024. Microbe/Phage Wastewater DNA/RNA Concentration and Extraction (Nanotrap® and NucleoMag® RNA Water). protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gpz8p5lzp/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: Working
We use this protocol and it's working
Created: April 05, 2024
Last Modified: April 05, 2024
Protocol Integer ID: 97861
Abstract
Wastewater epidemiology is a method used to study the diseases affecting a population. Conventional methods of wastewater collection for composite samples have involved the usage of autosamplers that require installation in sewers with appropriate expertise and certification. Tampon-based wastewater sampling is a cost-effective and simple way to collect wastewater to study microbial pathogens. It is also a safer alternative as tampons can be deployed and retrieved without opening manhole covers. In this protocol we detail a wastewater sampling strategy using tampons to obtain composite samples.
Next, wastewater-based epidemiology has predominantly been targeted, where scientists have looked for specific pathogens and use biomarkers for the same. Notably, traditional wastewater filtration methods (i.e. skim milk filtrations and membrane filtration) require large sample sizes with varying nucleic acid yields (Ahmed 2022). Recently, Nanotrap‱ technology has been shown to have a higher nucleic acid yield using a smaller sample size when compared to traditional membrane filtration techniques, making it a better tool for scientists to concentrate microbial nucleic acids in wastewater for wastewater epidemiology. Specifically, the Ceres Nanotrap‱ technology was developed to trace severe acute respiratory syndrome (SARS-CoV-2) in public wastewater and Nanotrap‱ particles have been used specifically to track viral pathogenic shedding in wastewater of: Endogenous pepper mottle virus (PMMoV), Influenza A, CrAssphage, and monkey pox. However, there is little known about the success rates of different downstream analysis techniques to concentrate microbes in wastewater in an untargeted fashion. Therefore, it remains unclear whether one method will work better than the other if one were to be interested in characterizing the microbial community composition and abundance in a wastewater sample. To this end, in this protocol we detail a wastewater processing method involving Ceres Nanotrap‱ technology and NucleoMag‱ RNA Water.
Virus Capture & Concentration
Virus Capture & Concentration
10m
Create a 1:100 dilution of Zoetis Bovine Rhinotracheitis-Parainfluzena-Respiratory Syncytial Virus (BRSV) Vaccine in H2O.
Collect 500 mL of wastewater in a 500mL or 1L plastic sample bottle.
500mL of wastewater collected in 500mL plastic bottles.
Spike the500 mL wastewater sample with 500 µL of 1:100 BSRV to achieve a 1µL/mL concentration of 1:100 BSRV.
Note
This can be applied to wastewater samples greater or less than 500mL, as long as a 1µL/mL concentration is achieved.
Mix the added 1:100 BRSV into the wastewater sample by aggressively inverting the bottle several times or shaking.
Incubate spiked wastewater samples for a minimum of 00:10:00 at room temperature (RT) to allow large aggregates to sediment at the bottom of the sample bottle.
10m
Use a 25mL serological pipette to transfer top 40 mL of spiked wastewater to a 50mL conical tube.
Note
Make sure to gently transfer from the top of the wastewater sample as to not disturb the sedimented particles at the bottom of the bottle.
Transferring 40mL of spiked and settled wastewater samples from 500mL bottles to 50mL conical tubes.
Add600 µL of Ceres Magnetic Nanotrap® particles to the 40 mL spiked wastewater aliquot.
Invert the wastewater samples several times to incorporate the Magnetic Nanotrap® particles and incubate for 20 minutes at RT.
Place conical tubes into custom magnet racks and allow magnets to attract Magnetic Nanotrap® particles for a minimum of 20 minutes at RT.
Note
The samples should be allowed to sit on the magnets long enough so that the supernatant is relatively clear. Some wastewater samples may be too turbid to reach this point, however.
Conical tubes with wastewater samples after incubation on magnets. Notice the rust-red Magnetic Nanotrap particles that have precipitated at the bottom of the tubes near the magnets.
Wastewater sample with Magnetic Nanotrap particles completely out of solution following magnet incubation.
Keeping the conical tubes fixed to the magnets, pour off the supernatant carefully as to not disturb the pellet of Magnetic Nanotrap® particles at the bottom of the conical tubes.
Viral RNA Extraction (Nucleomag® RNA Water)
Viral RNA Extraction (Nucleomag® RNA Water)
39m
Add 500 µL of Buffer MWA1 to the falcon tubes.
Vortex to resuspend the Magnetic Nanotrap® pellets in the Buffer MWA1.
Incubate samples for 00:10:00 at RT.
10m
Place conical tubes on the custom magnet racks to separate the Magnetic Nanotrap® particles.
With the tubes on the magnets, transfer 450 µL of lysate to a 2mL deep well plate.
Note
Use barcode labels on the falcon tubes to transfer sample ID's to a LIMS program when transferring lysate to the deep well plate.
Transferring lysate to a 2mL deep well plate for RNA extraction. Columns not being used in the current extraction run are sealed using non-pierceable foil.
Add 475 µL of Buffer MWA2 and 25 µL of NucleoMag® B-Beads to the lysate in the deep well plate.
Shake the sample plate for 00:05:00 at 1400 rpm , 56 °C .
5m
Place the sample plate on a plate magnet for at least 00:05:00 to separate NucleoMag® B-beads.
Note
At each magnet step, allow as much time as needed for the NucleoMag® B-beads to completely precipitate near the magnets.
5m
Use a multichannel pipette to remove the supernatant from each well.
Note
Ensure that NucleoMag® B-beads are not removed with the supernatant. If beads are drawn up with the supernatant, dispense the liquid back into the well and incubate on a magnet for a few minutes to allow the NucleoMag® B-beads to settle at the magnet.
Add850 µL of Buffer MWA3 to each well.
Shake the sample plate for 00:02:00 at 1400 rpm , 56 °C .
2m
Place the sample plate on a plate magnet for at least 00:02:00 to separate NucleoMag® B-beads.
2m
Use a multichannel pipette to remove the supernatant from each well.
Add850 µL of Buffer MWA3 to each well.
Shake the sample plate for 00:02:00 at 1400 rpm , 56 °C .
2m
Place the sample plate on a plate magnet for at least 00:02:00 to separate NucleoMag® B-beads.
2m
Use a multichannel pipette to remove the supernatant from each well.
Add850 µL of Buffer MWA4 to each well.
Shake the sample plate for 00:02:00 at 1400 rpm , 56 °C .
2m
Place the sample plate on a plate magnet for 00:02:00 to separate NucleoMag® B-beads.
2m
Use a multichannel pipette to remove the supernatant from each well.
Note
Take extra care to remove as much supernatant as possible at this step. Carryover of the wash buffer can cause PCR/qPCR/library prep to fail.
Place plate on shaker at 56 °C for approximately 30 minutes to allow beads to air dry.
Note
Ensure beads are thoroughly dry before proceeding, carry over EtOH from Buffer MWA4 can ruin downstream experiments.
Add 60 µL of RNase-free H2O to each well.
Shake the sample plate for 00:05:00 at 500 rpm , 56 °C .
5m
Place the sample plate on a plate magnet for 00:02:00 to separate NucleoMag® B-beads.
2m
Transfer eluted RNA to a 96-well elution plate for further processing.