TITLE: Automated concentration of viral particles in wastewater samples using Nanotrap Microbiome A Beads
AUTHORS: Ashlie McCunn, Dagmara Antkiewicz, Adelaide Roguet

[DESCRIPTION]
This method describes how to concentrate viral particles in water samples using magnetic beads (Nanotrap‱ Microbiome A Particles) to enable the subsequent quantification of microbial organisms in those samples. This method uses the KingFisherTM Apex automation system and may be used for wastewater, drinking water, surface and recreational waters.

[BEFORE_START]
Before and after using the BSC, clean work area with 10% bleach, followed by ELIMINaseTM, and 70% ethanol. Then, turn on the BSC interior ultraviolet (UV) light for at least 15 minutes. 
 












When using the benchtop, clean workspace area with 10% bleach, followed by ELIMINaseTM, and then 70% ethanol before and after use.
 


[GUIDELINES]
Wastewater samples should be stored at 4°C throughout processing. Prevent sample bottles from staying at room temperature for an extensive amount of time (10 minutes).
Remember to shake wastewater sample bottles thoroughly (at least 25 times) before processing the samples.

[STEPS]
SECTION: Concentration Preparation
4. For the Sample plates (Sample 1 and Sample 2): Inside the BSC, add 5 mL of wastewater sample into matching wells of the remaining two KingFisher Deep Well 24 Plates (10mL total are processed). Repeat this step for all samples listed on the plate map. 
 

SECTION: Concentration Preparation
4.1. Add 50 µL of Nanotrap Enhancement Reagent 2 to all wells in both Sample 1 and Sample 2 plates.
SECTION: Concentration: Loading KingFisher Apex
5. Go to the menu screen on the Thermo Scientific KingFisher Apex and select the program titled “NT Classic Extend 042822”.

SECTION: Concentration: Loading KingFisher Apex
5.2. Load the different plates according to the instructions on the screen. Ensure all 24-well plates are loaded with A1 corner in the upper right corner of the instrument's plate tray.

SECTION: Concentration: Loading KingFisher Apex
5.3. Lower the door and start the program. The program will run for 60 minutes.
 

 

SECTION: Concentration: Unloading KingFisher Apex
6. When the NT Classic Extend 042822 program is finished remove all plates from the Kingfisher Apex as instructed by the instrument prompts. 
SECTION: Concentration: Unloading KingFisher Apex
6.1. Discard Sample 1 plate, Sample 2 plate, and Tip plate.
SECTION: Concentration: Unloading KingFisher Apex
6.2. Keep the Elution plate for the next step.
SECTION: Concentrated Wastewater Samples
7. In the clean benchtop space, place the Elution plate on a Permagen 24-Well Ring Magnet Plate for 7-10 minutes to separate trace magnetic beads from the concentrated samples.
 

SECTION: Concentrated Wastewater Samples
7.1. Ensure magnetic beads have been separated from samples by looking for a solid ring around the base of each well (see photos below). Bead rings should be visible after sitting on magnetic separation plate for 7-10 minutes.
 

 

SECTION: Concentrated Wastewater Samples
7.2. Transfer 600 µL of sample from the elution plate (still sitting on the ring magnet plate) to its corresponding labeled storage tube. Be careful not to touch the tip of the pipette to the ring of magnetic beads at the base of the well. If the magnetic beads are transferred to the archive sample, they will interfere with the extraction process.

SECTION: Concentrated Wastewater Samples
7.3. Once all samples are removed, discard the Elution plate.

SECTION: Archive Storage
9. Store the concentrated wastewater samples in a freezer at -80 °C or below for up to 2 years.
SECTION: Sample Spike Prior to Concentration
1. Upon receipt, the temperatures of wastewater samples are measured and recorded. Samples with temperatures less than 2 °C and greater than 10 °C are recorded and flagged as temperatures outside of this range (2°C - 10°C) may compromise the sample quality. 
SECTION: Sample Spike Prior to Concentration
1.2. Store samples at 4 °C throughout processing.
SECTION: Concentration: Loading KingFisher Apex
5.1. Open the door of the KingFisher Apex, and click the next arrow to proceed.
 

SECTION: Sample Spike Prior to Concentration
1.1. Spike wastewater samples with 20 µL of bovine coronavirus vaccine (BCoV) per 250 mL of wastewater sample to serve as a virus recovery control measure at least 20 minutes before concentration. 
Separately, spike 50 mL of 10% Phosphate Buffered Saline (PBS) with 4 µL BCoV to serve as a method blank.

Sample receiving and spiking will take roughly 1 hour for 15-20 samples.
SECTION: Concentration Preparation
4.2. Remove Nanotrap Microbiome A Particles from refrigerator and vortex thoroughly (10-20 seconds). Avoid leaving magnetic particles at room temperature for an extensive amount of time (5 minutes).

SECTION: Extraction
8. Extraction of SARS-CoV-2 nucleic acid material may now be performed on the concentrated wastewater samples using any extraction method.
SECTION: Extraction
8.1. If not performing extraction on the concentrated samples, go to the next step.
SECTION: Concentration Preparation
4.3. Immediately after vortexing, add 75 µL of Nanotrap Microbiome A Particles to all wells with wastewater sample in both Sample 1 and Sample 2 plates.

SECTION: Concentration Preparation
2. Obtain three KingFisher Deep Well 24 Plates and one KingFisher Deep Well 24 Tip Plate.
SECTION: Concentration Preparation
3. For the Elution plate: On the benchtop, add 600 µL of   into each well of a KingFisher Deep Well 24 Plate as indicated by your plate map. Cover Elution plate with a Kim-wipe to avoid contamination and set aside to use later.