May 08, 2025
Ceres Nanotrap & Dynabeads Combined Wastewater Virus Enrichment & Total Nucleic Acid Extraction
- Victor Mabasa1,
- Natasha Singh1,
- Emmanuel Phalane1,
- Sipho Gwala1,
- Mokgaetji Macheke1,
- Thabo Mangena1,
- Lethabo Monametsi1,
- Lebohang Rabotapi1,
- Nkosenhle Ndlovu1,
- Fiona Els1,2,
- Sibonginkosi Maposa1,
- Said Rachida1,
- Kerrigan McCarthy1,3,
- Mukhlid Yousif1,3
- 1National Institute for Communicable Diseases;
- 2Gauteng City-Region Observatory;
- 3University of Witwatersrand
- Wastewater Genomics Syndicate
Protocol Citation: Victor Mabasa, Natasha Singh, Emmanuel Phalane, Sipho Gwala, Mokgaetji Macheke, Thabo Mangena, Lethabo Monametsi, Lebohang Rabotapi, Nkosenhle Ndlovu, Fiona Els, Sibonginkosi Maposa, Said Rachida, Kerrigan McCarthy, Mukhlid Yousif 2025. Ceres Nanotrap & Dynabeads Combined Wastewater Virus Enrichment & Total Nucleic Acid Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvjdoo5vk5/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: March 15, 2025
Last Modified: May 08, 2025
Protocol Integer ID: 124439
Keywords: wastewater ultra nucleic acid isolation kit, dynabeads combined wastewater virus enrichment, kingfisher flex purification system, total nucleic acid extraction this protocol, total nucleic acid extraction, nucleic acid extraction, dynabeads for virus enrichment, virus enrichment, purified nucleic acid, wastewater sample, ceres nanotrap bead, nucleic acid, ceres nanotrap, purification, extraction, applied biosystem, extraction process, digital pcr, pcr
Abstract
This protocol utilises Ceres Nanotrap beads and Dynabeads for virus enrichment from 20 mL wastewater samples. The Applied Biosystems MagMAX Wastewater Ultra Nucleic Acid Isolation Kit (Cat. No. A52610) is then employed for total nucleic acid extraction. The purified nucleic acids are suitable for various downstream applications, such as real-time PCR, digital PCR, and next-generation sequencing. Both the enrichment and extraction processes are automated on the KingFisher Flex Purification System with a 24-deep-well head, using in-house adapted programmes from MagMAX_Wastewater_10mL_Flex24.
Guidelines
General
- Perform all steps at room temperature (20–30°C), unless otherwise noted.
- Clean the work surfaces with RNaseZap to remove nucleases, then wipe the surfaces with 70% to 100% molecular biology grade ethanol to remove additional contaminants.
- Precipitates can form in the Lysis Buffer, Binding Solution, and Wash Buffer if stored below 20°C. If this occurs, warm the reagents at 37°C, then gently mix to dissolve the precipitates. Avoid creating bubbles.
Binding Bead Mix
- Vortex Binding Beads thoroughly before each use.
- Ensure that the beads stay fully mixed within the solution during pipetting.
- Avoid creating bubbles during mixing and aliquoting.
- The Binding Bead Mix is very dense so pipet carefully to ensure that the correct volume is added to the sample
Materials
Reagents
Nuclease-free water
Ceres Nanotrap Microbiome A Particles
Ceres Nanotrap Enhancement Reagent 1
100% Ethanol (molecular biology grade)
MagMAX Wastewater Ultra Nucleic Acid Isolation Kit (A52610)
Consumables
RNase-Free Microfuge Tubes, 1.5 mL
KingFisher Flex 24 Deep-Well Plate
MicroAmp Clear Adhesive Film
Conical Tubes (50 mL)
Equipment
KingFisher Flex Purification System with 24 deep‑well head
Standard laboratory vortex
Pipettes
Troubleshooting
Before start
- Prepare 80% ethanol using 100% absolute ethanol and nuclease-free water, ensuring a minimum volume of 2 mL per sample.
- Vortex the Binding Beads gently to ensure that the beads are fully resuspended.
- Prepare Binding Bead Mix — Combine 500 μL of Binding Solution with 20 μL of Binding Beads per sample, preparing enough for the required number of samples plus an additional 10% overage.
- Mix well by inversion, then store at room temperature.
Automated Dynabeads enrichment process
Set up and label your plates according to the following table:
| Plate name | Plate type | Contents | Vol per well | |
| Sample plate 1 | 24 deep-well | Clarified supernatant | 5 000 μL | |
| Dynabeads | 100 μL | |||
| Sample plate 2 | 24 deep-well | Clarified supernatant | 5 000 μL | |
| Lysis buffer | 24 deep-well | Lysis buffer | 500 μL | |
| Tip comb | 24 deep-well tip comb | Not applicable | ||
Select the appropriate program on the instrument (Wastewater_10mL_Virus_Isolation).
Start the run, then load the prepared sample and processing plates into position when prompted by the instrument.
While the instrument is running, set up the plates for Ceres Nanotrap enrichment process.
Automated Ceres Nanotrap enrichment process
Set up and label your plates according to the following table:
| Plate name | Plate type | Contents | Vol per well | |
| Sample plate 1 | 24 deep-well | Clarified supernatant | 5 000 μL | |
| Microbiome A particles | 75 μL | |||
| Enhancement Reagent 1 | 50 μL | |||
| Sample plate 2 | 24 deep-well | Clarified supernatant | 5 000 μL | |
| Microbiome A particles | 75 μL | |||
| Enhancement Reagent 1 | 50 μL | |||
| Lysis buffer | 24 deep-well | Lysis buffer | 500 μL | |
| Tip comb | 24 deep-well tip-comb |
Once the Dynabeads enrichment process is complete (~20 minutes after starting the run), remove the lysis plate from the instrument and store it at 4°C for nucleic acid extraction. Discard the remaining plates from the instrument.
Select the appropriate program (Wastewater_10mL_Virus_Isolation).
Start the run, then load the prepared Ceres Nanotrap enrichment plates into position when prompted by the instrument.
While the instrument is running, set up the nucleic acid extraction plates.
Once the Ceres Nanotrap enrichment process is complete (~20 minutes after starting the run), remove the lysis plate and store it at 4°C for nucleic acid extraction. Discard the remaining plates from the instrument.
Nucleic acid Extraction
Carefully combine 500 μL of lysed material from the Dynabeads plate with the corresponding 500 μL of lysed material from the Ceres Nanotrap plate.
Set up and label your plates according to the following table:
| Plate name | Plate type | Content | Vol per well | |
| Binding plate | 24 deep-well | Lysed sample | 1 000 μL | |
| Binding bead mix | 1 040 μL | |||
| Proteinase K | 80 μL | |||
| Wash buffer 1 | 24 deep-well | Wash buffer | 1 000 μL | |
| Wash buffer 2 | 24 deep-well | 80% Ethanol | 1 000 μL | |
| Elution buffer | 24 deep-well | Elution buffer | 100 μL | |
| Tip comb | 24 deep-well tip comb |
Load the Binding plate along with Wash 1, Wash 2, Elution, and a new tip comb. Start the run on the instrument and select the appropriate programme (Wastewater_10mL_Virus_Extraction)
Immediately remove the Elution plate from the instrument at the end of the run and cover it. Alternatively, transfer the eluate to a new tube or plate for nal storage. The isolated nucleic acid is ready for immediate use. For storage, keep it at –20°C for up to 6 months or at 80°C for longer than 6 months
Protocol references
This protocol is derived from Pub. No. MAN0025695 by Appliedbiosystems and a whitepaper by Ceres Nanotrap
Acknowledgements
This protocol is derived from Pub. No. MAN0025695 by Appliedbiosystems and a whitepaper by Ceres Nanotrap