Jul 19, 2024

Public workspaceWastewater grab sample processing with Nanotrap® Microbiome A particles (40 ml)

DOI
dx.doi.org/10.17504/protocols.io.x54v9px34g3e/v1
  • Dilip Abraham1,
  • Venkata Raghava Mohan2,
  • Nirmal Kumar1
  • 1Wellcome Trust Research Laboratory, Christian Medical College Vellore, India;
  • 2Department of Community Health and Development, Christian Medical College Vellore, India
Dilip Abraham
Open access
DOI: dx.doi.org/10.17504/protocols.io.x54v9px34g3e/v1
External link: https://www.ceresnano.com/_files/ugd/f7710c_c975f6fec8eb4183b3d8feb0a29041f8.pdf
Protocol Citation: Dilip Abraham, Venkata Raghava Mohan, Nirmal Kumar 2024. Wastewater grab sample processing with Nanotrap® Microbiome A particles (40 ml) . protocols.io https://dx.doi.org/10.17504/protocols.io.x54v9px34g3e/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: February 06, 2024
Last Modified: July 19, 2024
Protocol Integer ID: 94746
Keywords: Wastewater surveillance, sample processing, Nanotrap particles, wastewater concentration techniques
Funders Acknowledgement:
Bill & Melinda Gates Foundation
Grant ID: INV-049093
Abstract
This protocol is used to carry out processing of wastewater using Nanotrap‱ Microbiome A Particles (Ceres Nanosciences). Nanotrap‱particles are affinity-capture magnetic hydrogel particles that have been shown to capture and concentrate microorganisms (like SARS-CoV-2) from wastewater prior to RNA or TNA extraction. The sample is mixed with the particles, incubated, and separated with a magnet. The supernatant is removed, the pellet is spiked with extraction controls, and subjected to lysis followed by extraction. This protocol has been adapted from the online protocol available from Ceres Nanosciences.
Image Attribution
Barclay RA, Akhrymuk I, Patnaik A, et al. Hydrogel particles improve detection of SARS-CoV-2 RNA from multiple sample types. Sci Rep. 2020;10(1):22425. Published 2020 Dec 30. doi:10.1038/s41598-020-78771-8
Guidelines
Processed sample pellet needs to be extracted on the same day so as to avoid any nucleic acid degradation. The Nanotrap‱ A particles and the pellet obtained after sample concentration should not be frozen.
Materials
  1. ReagentNanotrap Magnetic Virus Particles (10)Ceres NanoCatalog #44202
  2. ReagentNanotrap Enhancer Reagent Er-2Ceres NanoCatalog #SKU 10112
  3. ReagentQIAamp® Fast DNA Stool Mini Kit QiagenCatalog #51604 - Inhibitex buffer
  4. ReagentqPCR DNA Extraction and Inhibition Control CY5-QXL670EurogentecCatalog #RT-SPCC-Q02
  5. ReagentRNA MS2 from Bacteriophage MS2RocheCatalog #10165948001
  6. ReagentNuclease-free water AmbionCatalog #AM9932
  7. Finnpipette F1 100 to 1000 μL Thermo Fisher Catalog #4641100N
  8. Finnpipette F1 20 to 200 μL Thermo Fisher Catalog #4641080N
  9. Finnpipette F1 2 to 20 μL Thermo Fisher Catalog #4641060N
  10. Finnpipette F1 0.2 to 2 μL Thermo Fisher Catalog #4641010N
  11. ART Barrier Specialty Pipette tips 1000 μL Thermo Fisher Catalog #2279-05PK
  12. ART Barrier Specialty Pipette tips 200 μL Thermo Fisher Catalog #2069-05PK
  13. ART Barrier Specialty Pipette tips 20 μL Thermo Fisher Catalog #2149P-05PK
  14. ART Barrier Specialty Pipette tips 10 μL Thermo Fisher Catalog #2139-05PK
  15. 50 mL Centrifuge Magnetic Stand 6 tube PERMAGEN- SKU# MSR6X50
  16. 1.5 mL Microcentrifuge Magnetic Stand 24 Tube PERMAGEN- SKU#MSR24
  17. 50 ml Centrifuge Tube with flat Cap, Sterile Pfact Catalog #PFACPCT-50-B-S
  18. 25 ml Serological Pipettes Nunc‱ Catalog #170368N
  19. 1.7 mL MaxyClear Snaplock Microcentrifuge Tube Axygen‱ Catalog #MCT-175-C
  20. Glass beads, acid-washed 212-300 μM (50- 70 U.S. sieve) Sigma Catalog #G1277-500G
  21. 2ml Screw Cap Micro Tubes Thermo Scientific‱ Catalog #346911
  22. 2 ml Snap Cap Low Retention Microcentrifuge Tubes Thermo Fisher Catalog #3453
Before start
Extraction control preparation
  • DNA control
Before first use of the sample process control (SPC), prepare a 1/10 dilution of the control DNA in nuclease free water and store them at Temperature-70 °C as small aliquots to avoid freeze thaw. Add Amount1 µL of 1/10 diluted SPCC into particle pellet.

  • RNA control
Prepare a 1/10 dilution of the MS2 extraction control in nuclease free water and store them at Temperature-70 °C as small aliquots to avoid freeze thaw. Add Amount1 µL of 1/10 diluted MS2 into particle pellet.
Wastewater grab sample processing with Nanotrap® Microbiome A particles
Wastewater grab sample processing with Nanotrap® Microbiome A particles
50m
Wastewater grab samples collected in Amount50 mL centrifuge tubes are transported to the laboratory in cold chain, maintaining a temperature of Temperature4 °C .
Once received, place the sample tubes on a tube rack and leave at TemperatureRoom temperature for 10 minutes, allowing large aggregates to settle at the bottom of the tube.
10m
Using a serological pipette, carefully transfer Amount40 mL of clear supernatant into a new 50 ml centrifuge tube without disturbing the sediment.
Critical
Add Amount400 µL of ER2 Enhancer reagent to the supernatant, invert 2-3 times and mix throughly.
Add Amount600 µL of Nanotrap‱ A particles to the above and invert mix 2-3 times. Incubate at TemperatureRoom temperature for Duration00:20:00 .
20m
Place the tube on a 50 ml magnetic stand for Duration00:10:00 min and allow the Nanotrap® particles to settle.
10m
Carefully discard the supernatant without disturbing the pellet. If needed, use a pipette to remove all the supernatant from the tube.
Remove the tube containing pellet from the magnetic rack. The pellet is now ready for total nucleic acid extraction. The pellet should not be frozen, extraction is to be carried out on the same day.
Critical
Add Amount700 µL of the InhibitEX buffer along with Amount1 µL of 1/10 diluted SPC and Amount1 µL of 1/10 diluted RNA-MS2 control into each particle pellet and resuspend the pellet by vortexing the tube briefly.
Transfer the suspension into a Amount1.7 mL microcentrifuge tube. Incubate at TemperatureRoom temperature for Duration00:10:00 .
10m
Place the microcentrifuge tubes on a 1.5 ml magnetic separation rack until the Nanotrap® particles have settled, resulting in a clear solution.
Collect the supernatant into Amount2 mL microcentrifuge tube containing ~ Amount370 mg of acid-washed glass beads and discard the pellet.
Protocol references
1. https://www.ceresnano.com/_files/ugd/f7710c_c975f6fec8eb4183b3d8feb0a29041f8.pdf

2. Barclay RA, Akhrymuk I, Patnaik A, et al. Hydrogel particles improve detection of SARS-CoV-2 RNA from multiple sample types. Sci Rep. 2020;10(1):22425. Published 2020 Dec 30. doi:10.1038/s41598-020-78771-8