TITLE: Nanotrap® KingFisher™ Concentration/Extraction &amp; MagMAX KingFisher™ Extraction
AUTHORS: Orlando   Sablon III, Jamie  VanTassell, Julia  Raymond, Marlene K Wolfe, Pengbo   Liu, Christine  Moe

[DESCRIPTION]
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.

[STEPS]
SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
1. 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.
 
  
SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
2. Aliquot 10 µL of Bovine Respiratory Syncytial Virus (BRSV)   to every well in Sample Plate 1 that will receive a wastewater sample.
 

SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
3. Aliquot 4865 µL of wastewater sample to one well (one well per sample) of Sample Plate 1 (the new KingFisher™ 24 Well Deep Well sample plate).
  

SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
4. Aliquot another 4865 µL of wastewater sample to the same well on Sample Plate 2 (a second KingFisher™ 24 Well Deep Well sample plate).

SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
5. Designate the same well on Sample Plate 1 and Sample Plate 2 as a negative control. Add 4865 µL of   into the designated well on both sample plates. 
 

SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
6. Aliquot 50 µL of  to each wastewater sample well, including the negative control well, in both sample plates.
 
SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
7. Incubate the sample plates for 10 min at 20 °C 
  
SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
8. Aliquot 75 µL of  to each wastewater sample well, including the negative control well, in both sample plates.
 

SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
9. Insert the KingFisher™ 24 well-KF Deep Well Comb into Sample Plate 1.
 

SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
4.1. 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.
  

SECTION: MagMAX KingFisher™ Extraction Procedure
14. Prepare Wash 1 Plate by aliquoting 1 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.
 


SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
10. Prepare the Lysis Plate by aliquoting 500 µL  of  to a third Kingfisher™ 24 Deep-Well Plate matching the number and location of the “Sample Plate” wells. 

SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
11. Run NT KingFisher™ Script
 

SECTION: Ceres Nanosciences Nanotrap® KingFisher™ Procedure
12. Once the protocol is completed, the KingFisher™ plate to which lysis solution was added will contain 500 µL of lysate that is ready to be run on the MagMAX KF extraction procedure.

SECTION: MagMAX KingFisher™ Extraction Procedure
15. Prepare the Elution Plate by aliquoting 60 µL of MagMAX Elution buffer to a new KingFisher™ 96 - 200 µL plate matching the number and location of the “Sample Plate” wells.

SECTION: MagMAX KingFisher™ Extraction Procedure
16. To prepare the Binding Plate, obtain a new KingFisher™ 96 Deep Well Plate.
SECTION: MagMAX KingFisher™ Extraction Procedure
17. Aliquot 10 µL of MagMAX Proteinase K to each well in the Binding Plate in which lysate will be added.

SECTION: MagMAX KingFisher™ Extraction Procedure
18. Aliquot 400 µL of the lysate from each well of the lysis plate used in Part 1 of the protocol into the Binding Plate.

 

SECTION: MagMAX KingFisher™ Extraction Procedure
19. Aliquot 530 µL of MagMAX Binding Solution into each deep well which lysate was added in the Binding Plate.

SECTION: MagMAX KingFisher™ Extraction Procedure
20. Vortex and thoroughly mix the MagMAX DNA/RNA Binding Beads. 
SECTION: MagMAX KingFisher™ Extraction Procedure
23. Prepare Wash Plate 2 by aliquoting 1 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.

SECTION: MagMAX KingFisher™ Extraction Procedure
22. Insert the KingFisher™ 96 Deep Well Comb into the Binding Plate.
SECTION: MagMAX KingFisher™ Extraction Procedure
21. Aliquot 20 µL of vortexed binding beads to each well in which lysate was added in the Binding Plate.
 


SECTION: MagMAX KingFisher™ Extraction Procedure
24. Run MagMAX KingFisher™ Protocol
 

SECTION: MagMAX KingFisher™ Extraction Procedure
25. 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. 

  

SECTION: MagMAX KingFisher™ Extraction Procedure
13. Label four KingFisher™ 96 Deep Well Plate-Sample Plate wells "Wash 1", "Binding", "Wash 2", and "Elution".