TITLE: Microbe/Phage Wastewater DNA/RNA Concentration and Extraction (Nanotrap® and NucleoMag® RNA Water)
AUTHORS: Brett Rasile, Kendra Maas, Archana Anand, Michael Hajkowski

[DESCRIPTION]
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. 

[STEPS]
SECTION: Virus Capture &amp; Concentration
2. Collect 500 mL of wastewater in a 500mL or 1L plastic sample bottle.
 

SECTION: Virus Capture &amp; Concentration
1. Create a 1:100 dilution of Zoetis Bovine Rhinotracheitis-Parainfluzena-Respiratory Syncytial Virus (BRSV) Vaccine in H2O.
 

SECTION: Virus Capture &amp; Concentration
3. Spike the500 mL wastewater sample with 500 µL of 1:100 BSRV to achieve a 1µL/mL concentration of 1:100 BSRV.
 



SECTION: Virus Capture &amp; Concentration
4. Mix the added 1:100 BRSV into the wastewater sample by aggressively inverting the bottle several times or shaking.
SECTION: Virus Capture &amp; Concentration
5. Incubate spiked wastewater samples for a minimum of 10 min at room temperature (RT) to allow large aggregates to sediment at the bottom of the sample bottle.


SECTION: Virus Capture &amp; Concentration
6. Use a 25mL serological pipette to transfer top 40 mL of spiked wastewater to a 50mL conical tube. 
 

 

SECTION: Virus Capture &amp; Concentration
7. Add600 µLof Ceres Magnetic Nanotrap® particles to the  40 mL spiked wastewater aliquot.

SECTION: Virus Capture &amp; Concentration
8. Place conical tubes into custom magnet racks and allow magnets to attract Magnetic Nanotrap® particles for a minimum of 20 minutes at RT.
 
 
 

SECTION: Virus Capture &amp; Concentration
9. 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.
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
10. Add 500 µL of Buffer MWA1 to the falcon tubes.

SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
10.1. Vortex to resuspend the Magnetic Nanotrap® pellets in the Buffer MWA1.
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
10.2. Incubate samples for 10 min at RT.

SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
11. Place conical tubes on the custom magnet racks to separate the Magnetic Nanotrap® particles.  

SECTION: Virus Capture &amp; Concentration
7.1. Invert the wastewater samples several times to incorporate the Magnetic Nanotrap® particles and incubate for 20 minutes at RT.
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
11.1. With the tubes on the magnets, transfer 450 µL of lysate to a 2mL deep well plate.
 
 

SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
12. Add 475 µL of Buffer MWA2 and 25 µL  of NucleoMag® B-Beads to the lysate in the deep well plate.

SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
12.1. Shake the sample plate for 5 min at   , 56 10 .

SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
12.2. Place the sample plate on a plate magnet for at least 5 min to separate NucleoMag® B-beads. 
 


SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
12.3. Use a multichannel pipette to remove the supernatant from each well.
 

SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
13. Add850 µL of Buffer MWA3 to each well.

SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
13.1. Shake the sample plate for 2 min at   , 56 10 .
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
13.2. Place the sample plate on a plate magnet for at least 2 min to separate NucleoMag® B-beads. 

SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
13.3. Use a multichannel pipette to remove the supernatant from each well.
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
14. Add850 µL of Buffer MWA3 to each well.
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
14.1. Shake the sample plate for 2 min at   , 56 10 .
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
14.2. Place the sample plate on a plate magnet for at least 2 min to separate NucleoMag® B-beads. 
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
14.3. Use a multichannel pipette to remove the supernatant from each well.
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
15. Add850 µL of Buffer MWA4 to each well.
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
15.1. Shake the sample plate for 2 min at   , 56 10 .
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
15.2. Place the sample plate on a plate magnet for 2 min to separate NucleoMag® B-beads. 
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
15.3. Use a multichannel pipette to remove the supernatant from each well.
 

SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
16. Place plate on shaker at 56 °C for approximately 30 minutes to allow beads to air dry.
 


SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
17. Add 60 µL of RNase-free H2O to each well.

SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
17.1. Shake the sample plate for 5 min at   , 56 10 .
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
17.2. Place the sample plate on a plate magnet for 2 min to separate NucleoMag® B-beads.
SECTION: Viral RNA Extraction (Nucleomag® RNA Water)
18. Transfer eluted RNA to a 96-well elution plate for further processing.