Jan 22, 2022

Public workspaceNanotrap® KingFisher™ Concentration/Extraction & MagMAX KingFisher™ Extraction

DOI
dx.doi.org/10.17504/protocols.io.b2nkqdcw
  • 1Center for Global Safe WASH, Rollins School of Public Health, Emory University, Atlanta GA USA
  • Emory University
Stephen P Hilton
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DOI: dx.doi.org/10.17504/protocols.io.b2nkqdcw
Protocol Citation: Orlando Sablon III, Jamie VanTassell, Julia Raymond, Marlene K Wolfe, Pengbo Liu, Christine Moe 2022. Nanotrap® KingFisher™ Concentration/Extraction & MagMAX KingFisher™ Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.b2nkqdcw
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: December 07, 2021
Last Modified: November 02, 2022
Protocol Integer ID: 55724
Keywords: Nanotrap, KingFisher, Ceres, nano, magnetic virus particles, magnetic, automated, wastewater, SARS-CoV-2, COVID-19
Funders Acknowledgements:
Ceres Nanosciences – NIH RADx Tech
Grant ID: 75N92021C00012
Abstract
These protocols have been adapted from Ceres "Nanotrap® Wastewater Protocol using MagMAX Kits" (APP-030, Revision 0, Nov 2021).

This protocol uses Nanotrap® Magnetic Virus Particles and Nanotrap® Enhancement Reagent 1 (ER1) to capture and concentrate viruses in wastewater samples. It is optimized for viral capture from 10 mL samples of wastewater and is compatible with three nucleic acid extraction kits from ThermoFisher. This protocol has been used and adapted for SARS-CoV-2 viral capture at Emory University for wastewater surveillance of COVID-19.
Materials
Autoclave - Amsco Lab 240 Steam Sterilizer
Bovine Respiratory Syncytial Virus (BRSV, INFORCE 3, Zoetis, Parsippany, NJ)(Control)
Corning Molecular Biology Grade Water (Reference no. 46-000-CM)
Wastewater sample
Pipettes
Eppendorf Research Plus Single Channel Pipette
Eppendorf Repeater Pipette
Ceres Nanosciences Nanotrap® Enhancement Reagent 1 (SKU 10111-10)
Ceres Nanosciences Nanotrap® Magnetic Virus Particles (In Solution) (SKU #44202)
Thermo Scientific™ KingFisher Apex
Thermo Scientific™ MagMAX™ Microbiome Lysis Solution (Catalog no. A42361)
Thermo Scientific™ MagMAX Viral/Pathogen Nucleic Acid Extraction Kit (ThermoFisher Cat# A42352):
  • Binding Buffer (Catalog no. A42359)
  • Proteinase K (Catalog no. A42363)
  • MagMAX Binding Beads (Catalog no. A42362)
  • Wash Buffer (Catalog no. A42360)
  • Elution Buffer (Catalog no. A42364)
80% Ethanol
Thermo Scientific™ KingFisher™ Plastics for 24 deep-well format (Catalog no. 95040470)
Thermo Scientific™ KingFisher™ Plastics for 24 deep-well tip comb and plate format (Catalog no. 97002610)
Thermo Scientific™ KingFisher™ Plastics for 96 deep-well format (Catalog no. 95040450)
Thermo Scientific™ KingFisher™ Plastics for 96 standard and PCR formats (Catalog no. 97002540)
Thermo Scientific™ KingFisher™ Plastics for 96 deep-well and tip comb format (Catalog no. 97002534)
Protocol materials
ReagentINFORCE 3 Intranasal Bovine VaccineZoetisCatalog #INF-00089
ReagentMolecular Biology Grade WaterCorningCatalog #46-000-CM
ReagentEnhancement Reagent 1Ceres NanoCatalog #10111-10
ReagentNanotrap Magnetic Virus Particles (10)Ceres NanoCatalog #44202
ReagentMagMAX™ Microbiome Lysis SolutionThermo FisherCatalog #A42361
Ceres Nanosciences Nanotrap® KingFisher™ Procedure
Ceres Nanosciences Nanotrap® KingFisher™ Procedure
10m
10m
Label two KingFisher™ 24 Well Deep Well sample plates “Sample Plate 1” and “Sample Plate 2”. You will split the total sample volume to process across the two plates for each sample.
Equipment
Plastics for 24 deep-well format
NAME
KingFisher
BRAND
95040470
SKU
https://www.thermofisher.com/order/catalog/product/95040470
LINK
Two Labeled KingFisher™ 24 Well Deep Well sample plates
Aliquot Amount10 µL of Bovine Respiratory Syncytial Virus (BRSV) ReagentINFORCE 3 Intranasal Bovine VaccineZoetisCatalog #INF-00089 to every well in Sample Plate 1 that will receive a wastewater sample.
Note
BRSV acts as a positive control and is only added to Sample Plate 1. Do NOT add BRSV to Sample Plate 2.

Aliquot Amount4865 µL of wastewater sample to one well (one well per sample) of Sample Plate 1 (the new KingFisher™ 24 Well Deep Well sample plate).
Wastewater sample is aliquoted into well A1 of Sample Plate 1.

Aliquot another Amount4865 µL of wastewater sample to the same well on Sample Plate 2 (a second KingFisher™ 24 Well Deep Well sample plate).

For example, if you loaded a sample into well A1 of Sample Plate 1, load the second volume of that sample into well A1 of Sample Plate 2.
Wastewater sample is aliquoted into well A1 of Sample Plate 2.

Designate the same well on Sample Plate 1 and Sample Plate 2 as a negative control. Add Amount4865 µL of ReagentMolecular Biology Grade WaterCorningCatalog #46-000-CM into the designated well on both sample plates.
Note
Do NOT introduce BRSV or wastewater into these two designated wells.

Aliquot Amount50 µL of ReagentEnhancement Reagent 1Ceres NanoCatalog #10111-10 to each wastewater sample well, including the negative control well, in both sample plates.
Incubate the sample plates for Duration00:10:00 at Temperature20 °C Room temperature
After adding Ceres Nanosciences Nanotrap® Enhancement Reagent 1, incubate both sample plates at room temperature for 10 minutes.
10m
Aliquot Amount75 µL of ReagentNanotrap Magnetic Virus Particles (10)Ceres NanoCatalog #44202 to each wastewater sample well, including the negative control well, in both sample plates.
Note
Prior to use, the product Nanotrap particles should be vortexed and thoroughly mixed.

Insert the KingFisher™ 24 well-KF Deep Well Comb into Sample Plate 1.
Equipment
Plastics for 24 deep-well tip comb and plate format
NAME
KingFisher
BRAND
97002610
SKU
https://www.thermofisher.com/order/catalog/product/97002610?SID=srch-hj-97002610
LINK

Prepare the Lysis Plate by aliquoting Amount500 µL of ReagentMagMAX™ Microbiome Lysis SolutionThermo FisherCatalog #A42361 to a third Kingfisher™ 24 Deep-Well Plate matching the number and location of the “Sample Plate” wells.

Run NT KingFisher™ Script
Note
  • If using a Kingfisher™ Flex System, run KF-008-WW-Nanotrap-24.bdz. If using
a Kingfisher™ Apex System, run KF-003-WW-Nanotrap-24.kfx.
  • Follow the on-screen instructions loading the previously prepared plates at the
appropriate time.

Once the protocol is completed, the KingFisher™ plate to which lysis solution was added will contain Amount500 µL of lysate that is ready to be run on the MagMAX KF extraction procedure.

MagMAX KingFisher™ Extraction Procedure
MagMAX KingFisher™ Extraction Procedure
Label four KingFisher™ 96 Deep Well Plate-Sample Plate wells "Wash 1", "Binding", "Wash 2", and "Elution".
During the MagMAX KingFisher™ Extraction Procedure, the following plates will be prepared: Wash 1, Binding, Wash 2, and Elution.

Prepare Wash 1 Plate by aliquoting Amount1 mL of MagMAX Wash Buffer to a new KingFisher™ 96 Deep Well Plate matching the number and location of the KingFisher™ 96 Deep Well Plate-Sample Plate wells.
Note
Applied Biosystems™ MagMAX™ Viral/Pathogen Nucleic Acid Isolation Kit contains:
  • Wash Buffer
  • Binding Buffer
  • Elution Buffer
  • Proteinase K
  • MagMAX Beads
Equipment
MagMAX™
NAME
Viral/Pathogen Nucleic Acid Isolation Kit
TYPE
Applied Biosystems™
BRAND
A42352
SKU
https://www.thermofisher.com/order/catalog/product/A42352?SID=srch-srp-A42352
LINK



Prepare the Elution Plate by aliquoting Amount60 µL of MagMAX Elution buffer to a new KingFisher™ 96 - 200 µL plate matching the number and location of the “Sample Plate” wells.

To prepare the Binding Plate, obtain a new KingFisher™ 96 Deep Well Plate.
Aliquot Amount10 µL of MagMAX Proteinase K to each well in the Binding Plate in which lysate will be added.

Aliquot Amount400 µL of the lysate from each well of the lysis plate used in Part 1 of the protocol into the Binding Plate.

Note
Keep track of which well contains which sample in this new bead binding plate. There should be about 100 μL of lysate remaining in each well of the lysis plate which can be discarded.

Aliquot Amount530 µL of MagMAX Binding Solution into each deep well which lysate was added in the Binding Plate.

Vortex and thoroughly mix the MagMAX DNA/RNA Binding Beads.
Aliquot Amount20 µL of vortexed binding beads to each well in which lysate was added in the Binding Plate.
Note
The total final volume should be 960 µL in each sample-containing well of this plate.  If volume exceeds 1000 µL, the possibility of contamination via spillage into adjacent wells can occur.


Insert the KingFisher™ 96 Deep Well Comb into the Binding Plate.
Prepare Wash Plate 2 by aliquoting Amount1 mL of 80% EtOH to a new KingFisher™ 96 Deep Well Plate matching the number and location of the KingFisher™ 96 Deep Well Plate-Sample Plate wells.

Run MagMAX KingFisher™ Protocol
Note
  • If using a Kingfisher™ Flex System, run KF-008-WW-MagMAX-96.bdz. If using
a Kingfisher™ Apex System, run KF-003-WW-MagMAX-96.kfx.
  • Follow the on-screen instructions loading the previously prepared plates at the
appropriate time.

Once the protocol is completed, the KingFisher™ 96-Elution Plate will contain ~60 µL purified viral RNA that is ready to be loaded onto a PCR plate or 1.7 mL RNA tube.

KingFisher™ 96-Elution Plate will contain ~60 µL purified viral RNA that is ready to be loaded onto a PCR plate or 1.7 mL RNA tube.