Nov 19, 2020
Automated high throughput viral concentration from wastewater using the KingFisher Flex platform
- 1University of California, San Diego
- Coronavirus Method Development Community
Protocol Citation: Smruthi Karthikeyan, Greg Humphrey 2020. Automated high throughput viral concentration from wastewater using the KingFisher Flex platform. protocols.io https://dx.doi.org/10.17504/protocols.io.bptemnje
Manuscript citation:
https://www.medrxiv.org/content/10.1101/2020.11.16.20232900v1
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: November 17, 2020
Last Modified: November 19, 2020
Protocol Integer ID: 44614
Keywords: WBE, high-throughput, viral concentration, wastewater
Abstract
Large-scale wastewater surveillance has the ability to greatly augment the tracking of infection dynamics especially in communities where the prevalence rates far exceed the testing capacity. However, current methods for viral detection in wastewater are severely lacking in terms of scaling up for high throughput. In the present study, we employed an automated magnetic-bead based concentration approach for viral detection in sewage that can effectively be scaled up for processing 24 samples in a single 40-minute run. The method compared favorably to conventionally used methods for viral wastewater concentrations with a limit of detection of 8.809 viral gene copies/ml from input sample volumes as low as 10ml and can enable the processing of over 100 wastewater samples in a day.
Protocol materials
Nanotrap Magnetic Virus Particles (10)Ceres NanoCatalog #44202
Step 2
MagMAX™ Microbiome Lysis SolutionThermo FisherCatalog #A42361
Step 3
MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit, with bead plateThermo FisherCatalog #A42357
Step 8
Aliquot 10 mL of raw sewage (no pre-filtering step required) into two wells of the KingFisher 24 deep-well plate (Cat# 95040470, Thermo Fisher). Each well should have 5 mL of the raw sewage sample. For instance, 5 mL of sample PL09 should be filled in A1 of plate 1 and 5 mL in A1 of plate 2.
Raw, unfiltered sewage
20m
Add 75 µL of Nanotrap Magnetic Virus Particles (10)Ceres NanoCatalog #44202 to each of the wells containing the raw sewage sample.
Plate 1: 5ml sample+ 75uL magnetic beads
5m
Prepare the elution plate: Add 150 µL of 1X PBS and 800 µL of theMagMAX™ Microbiome Lysis SolutionThermo FisherCatalog #A42361 to each of the wells of the 24 deep-well plate.
5m
Load the 3 plates into a KingFisher™ Flex Automated Nucleic Acid Purification system fitted with a 24 Deep-Well Head for KingFisher™ Flex Magnetic Particle Purification System (Cat. # 5400630 and 5400640).
Download the program for the KingFisher Platform for the automated processing of 24 samples:
NanoTrap_Flex24_V1ck.bdz and load the program on the the equipment using the BindIt Software (Cat. # 5189009).
NanoTrap_Flex24_V1ck.bdz and load the program on the the equipment using the BindIt Software (Cat. # 5189009). Once loaded, follow the steps on the the protocol to load the 3 plates into the system for extraction.
Note
The protocol provided here includes an extended incubation step which can be reduced by half depending on the sample volume. The above protocol can also be increased for processing 48 samples.
1h
The program processes the samples from the 2 sample plates and elutes the concentrated viral RNA into a single elution plate containing the lysis buffer.
Note
The Lysis buffer can vary depending on the Nucleic extraction kit used.
Nucleic acid extraction can then be performed using the MagMAX™ Microbiome Ultra Nucleic Acid Isolation Kit, with bead plateThermo FisherCatalog #A42357
Note
The downstream extraction can be performed with any kit of choice. Here the MagMAX kit was used due to ease of integration into the KingFisher Flex system.
36m